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Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of specific contractile regulatory proteins
Biochimica et Biophysica Acta 1449 (1999) 41^49
Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of speci¢c contractile regulatory proteins Arthur Beall
b;d
, Aaron Epstein
a;d
, David Woodrum
c;d
, Colleen M. Brophy
a;b;c;d;
*
a
b
Department of Surgery, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA Department of Medicine (Institute for Molecular Medicine and Genetics), Medical College of Georgia, Augusta, GA 30912, USA c Department of Cell Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA d Augusta Veterans Administration Medical Center, Augusta, GA 30901 USA Received 17 November 1998; accepted 23 November 1998
Abstract Cyclosporine A (CSA) is a type 2B phosphatase inhibitor which can induce contraction of renal artery smooth muscle. In this investigation, we examined the phosphorylation events associated with CSA-induced contraction of bovine renal artery smooth muscle. Contractile responses were determined in a muscle bath and the corresponding phosphorylation events were determined with whole cell phosphorylation and two-dimensional gel electrophoresis. CSA-induced contractions were associated with increases in the phosphorylation of the 20 kDa myosin light chains (MLC20 ) and different isoforms of the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions was associated with increases in the phosphorylation of another small heat shock protein, HSP20, and decreases in the phosphorylation of the MLC20 , and some isoforms of HSP27. These data suggest that CSA-induced contraction and relaxation of vascular smooth muscle is associated with increases in the phosphorylation of specific contractile regulatory proteins. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Cyclosporine; Smooth muscle, vascular; Myosin light chain; Heat shock protein
1. Introduction Cyclosporine A (CSA) is an immunosuppressant drug used for the prevention of graft rejection after transplantation. The clinical usefulness of CSA is limited by vascular toxicity, which is manifest by systemic hypertension and nephrotoxicity [1,2]. CSA therapy leads to an increase in renal vascular
* Corresponding author, at address a. Fax: (706) 8233949; E-mail: [email protected]
resistance and a decrease in renal blood £ow in humans and in animal models [3,4]. It has been proposed that CSA decreases renal blood £ow by inducing the release of vasoconstrictor substances, decreasing the endothelial production of vasorelaxants, or by a direct e¡ect on the vascular smooth muscle [5^8]. The e¡ects of CSA on the vascular smooth muscle include enhanced responsiveness to vasoconstrictors, decreased responsiveness to vasorelaxants, and a direct contractile e¡ect on rat aortic and bovine renal smooth muscle [6,8^11]. The proposed mechanism of action of CSA as an
0167-4889 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 9 8 ) 0 0 1 6 9 - 4
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immunosuppressant is via binding to the intracellular cyclophilins and inactivating the Ca2 - and calmodulin-dependent protein phosphatase (type 2B phosphatase otherwise known as calcineurin) in T-lymphocytes [12]. Since other phosphatase inhibitors, such as okadaic acid and calyculin, are potent mediators of vascular smooth muscle contraction [13,14], we hypothesized that CSA-induced vascular smooth muscle contractions would be associated with increases in the phosphorylation of speci¢c contractile regulatory proteins. 2. Materials and methods 2.1. Materials CSA was from Sandoz (East Hanover, NJ); [ P]orthophosphate from Amersham (Arlington Heights, IL); N-2-hydroxyethylpiperazine-NP-2-ethanesulfonic acid (HEPES), ethylene glycol-bis(L-amino ethyl ether) N,N,NP,NP-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), cremophore, and all other analytical grade chemicals were from Sigma (St. Louis, MO). Mouse monoclonal anti-HSP27 antibodies were from Dr. M.J. Welsh (University of Michigan, Ann Arbor, MI) [15] and rabbit polyclonal anti-MLC20 antibodies were from Dr. J. Stull (University of Texas, Dallas, TX). Goat anti-mouse secondary antibodies were from Jackson Immunochemical (West Grove, PA). Electrophoresis reagents and the DC protein assay kit were from Bio-Rad (Hercules, CA). 32
2.2. Muscle bath experiments Bovine renal arteries were obtained from an abattoir and placed directly in cold HEPES bu¡er (140 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4 , 1.0 mM NaH2 PO4 , 1.5 mM CaCl2 , 10 mM glucose, and 10 mM HEPES, pH 7.4). The vessels were opened longitudinally and the adventitia was dissected free of the vessel. The endothelium was removed by rubbing the strip with a cotton tipped applicator. Transverse strips, 1.0 mM in diameter, were cut, and a loop of 3-0 silk was tied to each end. The strips were suspended in a muscle bath containing bicarbonate bu¡-
er (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4 , 1.0 mM NaH2 PO4 , 10 mM glucose, 1.5 mM CaCl2 , and 25 mM Na2 HCO3 , pH 7.4), equilibrated with 95% O2 /5% CO2 , at 37³C for at least 1 h. Changes in isometric force were registered by tension transducers (Grass, Quincy, MA) and recorded on a chart recorder (Gould, Norcross, GA). The strips were progressively stretched and the isometric force generated in response to 110 mM KCl (made with equimolar replacement of NaCl in bicarbonate bu¡er) was determined until optimal tension was produced. CSA and forskolin were solubilized in DMSO and then added directly to the muscle bath at dilutions greater than 1/100. DMSO, 1/100 dilution, had no e¡ect on smooth muscle contractile responses (data not shown). 2.3. Whole cell phosphorylation and two-dimensional gel electrophoresis Strips of renal artery smooth muscle were equilibrated in bicarbonate bu¡er bubbled with 95% O2 / 5% CO2 for 1 h at 37³C. The strips were then rinsed and incubated in low-phosphate bu¡er (10 mM HEPES pH 7.4, 140 mM NaCl, 4.7 mM KCl, 1.0 mM MgCl2 , 1.5 mM CaCl2 , 10 mM glucose, and 0.3 mM NaH2 PO4 ) for 30 min. The strips were then equilibrated in low-phosphate bu¡er containing 250 WCi/ml [32 P]orthophosphate for 1 h. The strips were treated with CSA (0.4 mM for 10 min) or remained in bu¡er as controls. The doses of CSA used in this study were the half-maximal dose of CSA required for contraction of bovine renal artery smooth muscle in vitro [11]. The incubation was terminated by immersing the muscle strips in a dry iceacetone slurry and then crushing the tissue with mortar and pestle under liquid N2 . The powder was resuspended in homogenization bu¡er (20 mM HEPES pH 7.4, 20 mM sucrose, 100 mM NaF, 15 mM EDTA, 2 mM EGTA, 1 mM PMSF, and 1.3% SDS) and boiled for 5 min. Two-dimensional gel electrophoresis was performed using vertical slab isoelectric focusing gels with the modi¢cation described by Hochstrasser et al. [16]. The conditions were optimized to emphasize the low molecular weight phosphoproteins (in the range of MLC20 ). Brie£y, the samples were acetone-precipitated and reconstituted in 9 M urea and
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2% CHAPS. The protein concentrations of the samples were determined and 150 Wg of protein were adjusted to a ¢nal concentration of 9 M urea, 2% CHAPS, 100 mM dithiothreitol (DTT), 15% glycerol, and 5% ampholines (5 parts 6^8, 3 parts 5^7, 2 parts 3^10). The ¢rst dimensions were focused for 10 000 Vh, and then run on a 12% SDS-PAGE second dimension [17]. The gels were stained with Coomassie brilliant blue, dried, and exposed to Kodak XAR ¢lm. For immunoblotting, second dimension PAGE gels were transferred to Immobilon-P (Millipore, Bedford, MA), blocked with TBS-milk (10 mM Tris (pH 7.4), 150 mM NaCl, 5% milk), washed with TBS-Tween (10 mM Tris (pH 7.4), 150 mM NaCl, 0.5% Tween 20), and probed with mouse anti-HSP27 antibody (1:5000 in TBS-milk for 1 h). Immunoreactive proteins were detected using peroxidase conjugated goat anti-mouse (1:2000 in block bu¡er) and Western blot chemiluminescence reagent (DuPont-NEN, Boston, MA).
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non-phosphorylated MLC20 were quantitated with a PhosphorImager. 2.5. Statistical analysis Values are reported as mean þ S.E.M. and n refers to the number of animals examined. The statistical di¡erences between groups were determined with one way repeated measures analysis of variance (ANOVA) using Sigma Stat software (Jandel Scienti¢c, San Rafael, CA). A P value less than 0.05 was considered signi¢cant. Densitometric analysis of immunoreactive HSP27 was performed using a CCD camera interfaced with a 2D analyzer software package (Innovision Software, Bioimage, Ann Arbor, MI). The MLC20 bands and phosphorylated proteins on two-dimensional gels were quantitated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software (Molecular Dynamics).
2.4. MLC20 phosphorylation Strips of bovine renal artery smooth muscle were equilibrated in a muscle bath as described above. The strips were kept in bu¡er (control) or stimulated with KCl (110 mM) or CSA (0.4 mM). When a maximal contractile response was obtained (after 10 min), the muscles were immediately snap frozen with liquid N2 cooled tongs and ground to a ¢ne powder under liquid N2 . The powder was placed in 90% acetone, 10% trichloroacetic acid, 10 mM DTT and subsequently washed 3 times with 100% acetone, 10 mM DTT. The pellet was resuspended in 9 M urea, 2% CHAPS, 100 mM DTT and protein concentrations determined. Twenty Wg of protein was separated on glycerol-urea mini-gels (40% glycerol, 15% acrylamide, 0.75% bisacrylamide, 10 mM Tris, and 22 mM glycine) at 150 V for 19 h in the cold room [18]. The proteins were then transferred to Immobilon (Millipore, Bedford, MA), 100 V for 1 h. The blots were blocked with TBS-milk and then incubated overnight with anti-MLC20 antibodies (1:4000 in TBS-milk). After washing (6 times with TBS-Tween), the blots were incubated in 125 I-protein A and the relative amounts of phosphorylated and
Fig. 1. CSA-induced contractions of renal artery smooth muscle. Strips of renal artery smooth muscle were equilibrated in a muscle bath and after the initial response to high extracellular KCl (110 mM) was determined, the strips were contracted with CSA (0.4 mM). The CSA was then washed out (W, panel A), or remained in the bath and sodium nitroprusside (10 WM, S, panel B), or forskolin (10 WM, F, panel C) was added.
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centrations (1500^2000 ng/ml) that can occur in clinical settings.
3. Results 3.1. CSA is a direct vascular smooth muscle contractile agonist CSA (0.4 mM) induces contraction in bovine renal artery smooth muscles which relax when the CSA was washed out (100% relaxation, n = 6, Fig. 1); or with the addition of the vasorelaxants, sodium nitroprusside (10 WM, 94 þ 3% relaxation, n = 8, Fig. 1); or forskolin (10 WM, 88 þ 6% relaxation, n = 6, Fig. 1). The dose of CSA used in this study represents the dose necessary for half-maximal contraction of bovine renal artery smooth muscle [11]. This dose is higher than optimal serum levels of CSA (250^350 ng/ml); however, it approaches the peak serum con-
3.2. Phosphorylation events associated with CSA-induced contractions in renal artery smooth muscle Whole cell phosphorylation and two-dimensional gel electrophoresis of strips of renal artery smooth muscles treated with CSA (0.4 mM for 10 min) led to increases in the phosphorylation of speci¢c phosphoproteins. There were increases in the phosphorylation of multiple 27 kDa proteins with di¡erent isoelectric points (Fig. 2, Table 1). Bitar and coworkers have suggested that phosphorylation of the 27 kDa small heat shock protein, HSP27, is involved in sustained
Fig. 2. CSA-induced phosphorylation changes in bovine renal artery smooth muscle. Strips of bovine renal artery smooth muscle were loaded with [32 P]orthophosphate and equilibrated in bu¡er (panels A, D, G), treated with cyclosporine A (CSA, 0.4 mM, for 10 min, panels B, E, H) or treated with cyclosporine A (0.4 mM for 10 min) followed by forskolin (10 WM for 10 min, panels C, F, I). The strips were homogenized and the proteins separated by isoelectric focusing in the ¢rst dimension (indicated by pI 4.5^6.5) and SDSPAGE in the second (the relative mobility of the proteins is indicated by the molecular weight markers on the left of the gels). The proteins were transferred to Immobilon and the membrane was developed with a PhosphorImager (autoradiographs are depicted in panels A, B, C). The membranes were then probed with anti-HSP27 antibodies (panels D, E, F) and re-probed with anti-HSP20 antibodies (panels G, H, I). The location of the myosin light chains (M), the isoforms of HSP27 (A, B, C), and the isoforms of HSP20 (Nos. 8, 3, 4, as previously labeled by Takuwa [22]) are indicated on the blots. Non-phosphorylated, immunoreactive HSP20 and HSP27 are also indicated. These blots are representative of ¢ve separate experiments.
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Table 1 Quantitation of phosphorylation changes induced by CSA alone and CSA followed by forskolin in bovine renal artery smooth muscle Protein
CSA
CSA/FSK
Reference MLC20 HSP27A HSP27B HSP27C HSP20 #4 HSP20 #3 HSP20 #8
1.07 þ 0.08 2.14 þ 0.65* 1.76 þ 0.26* 1.78 þ 0.19* 1.83 þ 0.21* 2.01 þ 0.14* 1.35 þ 0.21 2.06 þ 0.90*
1.02 þ 0.13 0.72 þ 0.27 1.32 þ 0.26 1.29 þ 0.22 1.76 þ 0.51* 2.26 þ 0.53* 2.40 þ 0.33* 5.40 þ 0.60*
The values are the mean of the fold increase over control þ S.E.M. for cyclosporine (CSA, 0.4 mM, 10 min) and CSA (0.4 mM, 10 min) followed by forskolin (FSK, 10 WM, 10 min) treated strips (n = 5, *P 6 0.05, ANOVA compared to control). `Reference' refers to the same reference protein identi¢ed by Takuwa et al. [22]; HSP27A is the isoform of HSP27 with a pI of 5.5; HSP27B, pI 5.7; HSP27C, pI 5.9; HSP20 #4 is the isoform of HSP20 with a pI of 6.0; HSP20 #3, pI 5.9; and HSP20 #8, pI 5.7.
smooth muscle contraction [18]. To determine whether the 27 kDa proteins, which demonstrated an increase in phosphorylation after CSA treatment were HSP27, two-dimensional gels were transferred
Fig. 4. CSA treatment leads to increases in the phosphorylation of MLC20 . Strips of bovine renal artery smooth muscle were equilibrated in a muscle bath and after an initial high KCl contraction, remained in bu¡er (CONT), or were treated with high KCl (110 mM, for 5 min), or CSA (0.4 mM, 10 min). The samples were separated on glycerol-urea gels [18], and probed with antibodies against MLC20 . A representative blot is depicted in panel A and the arrow identi¢es the phosphorylated isoform of MLC20 . Densitometric analysis of multiple blots is depicted in panel B (n = 5 di¡erent strips, error bars depict standard error of the mean, *P 6 0.05, ANOVA).
Fig. 3. CSA treatment leads to increases in the immunoreactive isoform of HSP27 in bovine renal artery smooth muscle. Strips of bovine renal artery smooth muscle were equilibrated as controls (CONT) or treated with CSA (0.4 mM for 10 min) and the proteins were separated on two-dimensional gels. The proteins were transferred to Immobilon and probed with antiHSP27 antibodies. The relative density of the spots was quantitated as described in the speci¢c methods and the fraction of the immunoreactive acidic isoform of HSP27 (isoform A) expressed as a percentage of the sum of the total immunoreactive HSP27 (n = 5, error bars indicate standard error of the mean, *P 6 0.05 ANOVA).
to Immobilon and probed with anti-HSP27 antibodies. Proteins immunoreactive with anti-HSP27 antibodies demonstrated a similar mobility on two-dimensional gels as the 27 kDa phosphoproteins (Fig. 2). There was an immunoreactive spot with a relative mobility of 27 kDa which did not correspond to a phosphorylated 27 kDa protein. This likely represents non-phosphorylated HSP27. Densitometric analysis of the immunoblots demonstrated a signi¢cant increase in the amount of the most acidic isoform (isoform A) of HSP27 as a fraction of the total immunoreactive HSP27 after CSA treatment (Fig. 3). CSA treatment (0.4 mM for 10 min) also led to increases in the phosphorylation of the MLC20 . Proteins from muscles treated with or without CSA (0.4 mM, for 10 min) were separated on glycerol-urea gels and probed with antibodies against MLC20 [19]. Proteins from muscles treated with high KCl were used as positive controls. Treatment with CSA led to increases in the phosphorylation of the MLC20 that
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were similar to the increases that occurred with high extracellular KCl (Fig. 4). Forskolin-induced relaxation of renal artery smooth muscles that had been pre-contracted with CSA led to decreases in the phosphorylation of the MLC20 some isoforms of HSP27 (Table 1). Cyclic nucleotide-dependent relaxation was associated with increases in the phosphorylation of three 20 kDa proteins (Table 1). These proteins have recently been identi¢ed as di¡erent isoforms of the small heat shock protein, HSP20 [20]. Proteins immunoreactive with anti-HSP20 antibodies demonstrated a similar mobility on two-dimensional gels as the 20 kDa phosphoproteins (Fig. 2). 4. Discussion Chronic administration of CSA signi¢cantly and reproducibly produces hypertension in humans and animal models [1]. This may be due to a direct e¡ect of CSA on the vascular smooth muscle [8,11]. Increases in the phosphorylation of a number of proteins including the myosin light chains (MLC20 ), desmin, caldesmon, and the small heat shock protein, HSP27, have been associated with the contractile state of vascular smooth muscles [21^23]. Increases in the phosphorylation of these proteins may occur with activation of speci¢c kinases which are capable of phosphorylating the speci¢c proteins, or inhibition of the phosphatases which de-phosphorylate these proteins [24]. CSA is a speci¢c inhibitor of Ca2 / calmodulin-dependent phosphatase (phosphatase 2B or calcineurin) [12]. Inhibitors of type 1 and 2A phosphatases have been shown to directly induce vascular smooth muscle contraction, presumably as a result of an inhibition of the de-phosphorylation of speci¢c proteins [13,14]. However, the e¡ect of CSA, a type 2B phosphatase inhibitor, on protein phosphorylation events in vascular smooth muscles has not been previously examined. In this investigation we determined that CSA-induced contractions are associated with increases in the phosphorylation of a set of speci¢c proteins similar to those that become phosphorylated during agonist-induced contractions in multiple types of smooth muscles [21^23]. Stimulation of renal artery smooth muscle with CSA led to increases in the
phosphorylation of MLC20 (Table 1, Fig. 4). Increases in the phosphorylation of the MLC20 are thought to regulate the initiation of smooth muscle contraction [25]. However, sustained vascular smooth muscle contraction has been described in many systems in which the state of MLC20 phosphorylation returns to its baseline value during the sustained phase of contraction [26]. Thus, numerous investigators have implicated other phosphorylation events in mediating the sustained phase of contraction [27^29]. CSA treatment also led to increases in the phosphorylation of the small heat shock protein, HSP27 (Table 1, Figs. 2 and 3). Other investigators have noted increases in the phosphorylation of 27 kDa proteins that is temporally associated with smooth muscle contraction [23]. Increases in the phosphorylation of HSP27 is also associated with carbachol stimulated tracheal smooth muscle contraction [30] and thrombin-induced vascular smooth muscle contractions [31]. Prolonged treatment of animals with CSA leads to an attenuation of the endothelial-independent relaxation response to sodium nitroprusside [10]. In addition, vascular smooth muscle contractions induced by the type I/IIa phosphatase inhibitor, calyculin, are refractory to relaxation with the addition of sodium nitroprusside [32]. However, contractions induced by the acute administration of CSA in a muscle bath relax rapidly with the addition of the guanylate cyclase activator, sodium nitroprusside or the adenylate cyclase activator, forskolin (Fig. 1). Cyclic nucleotide-dependent relaxation of CSA-precontracted renal artery smooth muscles is associated with decreases in the phosphorylation of the MLC20 and some isoforms of HSP27 (Table 1). Relaxation was also associated with increases in the phosphorylation of two 20 kDa proteins that have recently been identi¢ed as HSP20 (Fig. 2, Table 1) [20]. Heat shock proteins function as molecular chaperones which confer resistance to cellular stresses. While the function of HSPs has been well established in cultured cells, less is known about the actual physiologic functions of HSPs in intact tissues. These data and the data of others suggest that the small HSPs may play a role in modulating the contractile state of smooth muscles. Bitar and co-workers have recently implicated HSP27 as a mediator of sustained
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rectal sphincter smooth muscle tone [18]. HSP27 has been shown to be relatively abundant in muscle tissue [33] and recent data suggest that HSP27 interacts with and regulates the actin cytoskeleton [34^37]. HSP20 was initially identi¢ed as a byproduct of the puri¢cation of HSP27 and increases in the phosphorylation of HSP20 are associated with cyclic nucleotide-dependent relaxation of smooth muscle [20,38]. The small HSPs exist intracellularly as large macromolecular aggregates [39^42]. Thus, it is possible that the oligomeric association of HSP27 and HSP20 functions as a molecular chaperone to stabilize the contractile machinery resulting in sustained contraction and that phosphorylation of HSP20 disrupts this interaction leading to relaxation. Several phosphatases have been shown to use phosphorylated MLC20 as a substrate in vitro and the majority of these phosphatases have been classi¢ed as type 1 or 2A. A myosin bound phosphatase has been identi¢ed that is a trimeric type 1 enzyme [43]. Increases in the phosphorylation of MLC20 in vascular smooth muscle treated with CSA suggest that type 2B phosphatase may also regulate MLC20 phosphorylation. The Ca2 /calmodulin-dependent protein phosphatase (calcineurin) has been implicated in the de-phosphorylation of HSP25, the murine analogue of bovine and human HSP27 [44]. Since the activation of many kinases is dependent on phosphorylation, it is also possible that the increases in phosphorylation of speci¢c smooth muscle proteins associated with CSA treatment are due to an inhibition of the de-phosphorylation of speci¢c kinases leading to persistent activation of the kinases. In summary, CSA-induced renal artery smooth muscle contractions are associated with increases in the phosphorylation of a similar speci¢c set of proteins as occurs with agonist-induced contraction. One of these proteins was identi¢ed as the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions are associated with decreases in the phosphorylation of these regulatory proteins and with increases in the phosphorylation of the small heat shock protein, HSP20. CSA may directly induce vascular smooth muscle contraction by speci¢cally inhibiting the de-phosphorylation of smooth muscle regulatory proteins or by inhibiting the de-phosphorylation of kinases thus producing persistent activation of the kinases.
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Acknowledgements This work was supported by a grant from the VA Merit Review, the NIH (1RO1HL58027-02), and an American Heart Association Clinician Scientist Award (to Dr. C.M. Brophy). The authors appreciate the assistance of Shapiro's Meatpacking Corp. for donating bovine blood vessels. The authors also appreciate the donation of anti-MLC20 antibodies from Dr. James Stull and anti-HSP27 antibodies from Dr. Michael Welsh. References [1] D.V. Hamilton, D.J.S. Carmichael, D.B. Evans, R.Y. Calne, Hypertension in renal transplant recipients on cyclosporine A and corticosteroids and azathioprine, Transplant. Proc. 14 (1982) 597^600. [2] B.D. Myers, J. Ross, L. Newton, L. Luetscher, M. Perlroth, Cyclosporine-associated chronic nephropathy, New Engl. J. Med. 311 (1984) 699^705. [3] J. English, A. Evan, D.C. Houghton, W.M. Bennett, Cyclosporine-induced renal dysfunction in the rat, Transplantation 44 (1987) 135^141. [4] M.R. Weir, D.K. Klassen, S.Y. Shen, D. Sullivan, E.U. Buddenmeyer, B.S. Handwerger, Acute e¡ects of intravenous cyclosporine on blood pressure, renal hemodynamics, and urine prostaglandin production of healthy humans, Transplantation 49 (1990) 41^47. [5] D.M. Lanese, J.D. Conger, E¡ects of endothelin receptor antagonist on cyclosporine-induced vasoconstriction in isolated rat renal arterioles, J. Clin. Invest. 91 (1993) 2144^ 2149. [6] A. Rego, R. Vargas, M.L. Foegh, P.W. Ramwell, E¡ect of cyclosporine A treatment on vascular reactivity of the rat thoracic aorta, Transplant. Proc. 20 (1988) 572^577. [7] K. Sudir, J.S. MacGregor, T. DeMarco, C.J.M. DeGroot, R.N. Taylor, T.M. Chou, P.G. Yock, K. Chatterjee, Cyclosporine impairs release of endothelium-derived relaxing factors in epicardial and resistance coronary arteries, Circulation 90 (1994) 3018^3023. [8] H. Xue, R.D. Bukowki, D.A. McCarron, W.M. Bennet, Induction of contraction in isolated rat aorta by cyclosporine, Transplantation 43 (1987) 715^718. [9] M.D. Gar, M.S. Paller, Cyclosporine augments renal but not systemic vascular reactivity, Am. J. Physiol. 258 (1990) F211^F217. [10] H.C. Huang, A. Rego, R. Vargas, M.D. Foegh, P.W. Ramwell, Nitroprusside-induced vascular relaxation is attenuated in organ-transplanted animals treated with cyclosporine, Transplant. Proc. 19 (1987) 126^130. [11] A. Epstein, J. Wynn, L. Mulloy, C.M. Brophy, Cyclosporine, but not FK506, selectively induces renal and coronary
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and active tension during muscarinic stimulation of tracheal smooth muscle, J. Pharmacol. Exp. Ther. 240 (1987) 8^ 15. H. Rasmussen, Y. Takuwa, S. Park, Protein kinase C in the regulation of smooth muscle contraction, FASEB 1 (1987) 177^185. H. Katsuyama, K. Morgan, Mechanisms of calcium independent contraction in single permeabilized ferret aorta cells, Circ. Res. 72 (1993) 651^657. G. Whitney, D. Throckmorton, C. Isales, Y. Takuwa, J. Yeh, H. Rasmussen, C. Brophy, Kinase activation and smooth muscle contraction in the presence and absence of calcium, J. Vasc. Surg. 22 (1995) 37^44. J.K. Larsen, I.A. Yamboliev, L.A. Weber, W.T. Gertho¡er, Phosphorylation of the 27-kDa heat shock protein via p38 MAP kinase and MAPKAP kinase in smooth muscle, Am. J. Physiol. 273 (1997) L930^L940. C.M. Brophy, D. Woodrum, M. Dickinson, A. Beall, Thrombin activates MAPKAP2 kinase in vascular smooth muscle, J. Vasc. Surg. 27 (1998) 963^969. C.M. Brophy, E.G. Whitney, S. Lamb, A. Beall, The cellular mechanisms of nitric oxide induced vasorelaxation, J. Vasc. Surg. 25 (1997) 390^397. Y. Inaguma, S. Goto, H. Shinohara, K. Hasegawa, K. Ohshima, K. Kato, Physiological and pathological changes in levels of the two small stress proteins, HSP27 and aB crystallin, in rat hindlimb muscles, J. Biochem. 114 (1993) 378^ 384. J.N. Lavoie, E. Hickey, L.A. Weber, J. Landry, Modulation of actin micro¢lament dynamics and £uid phase pinocytosis by phosphorylation of heat shock protein 27, J. Biol. Chem. 268 (1993) 24210^24214. B.G. Leicht, H. Biessmann, K.B. Palter, J.J. Bonner, Small heat shock proteins of Drosophila associate with the cytoskeleton, Proc. Natl. Acad. Sci. USA 83 (1986) 90^94. T. Miron, M. Wilchek, B. Geiger, Characterization of an inhibitor of actin polymerization in vinculin-rich fraction of turkey gizzard smooth muscle, Eur. J. Biochem. 178 (1988) 543^553. T. Miron, K. Vancompernoll, J. Vanderkerckhove, M. Wilchek, B. Geiger, A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein, J. Cell Biol. 114 (1991) 255^261. K. Kato, S. Goto, Y. Inaguma, K. Hasegawa, K. Morishita, T. Asano, Puri¢cation and characterization of a 2-kDa protein that is highly homologous to aB-Crystallin, J. Biol. Chem. 269 (1994) 15302^15309. P. Mehlen, A. Arrigo, The serum-induced phosphorylation of mammalian hsp27 correlates with changes in its intracellular localization and levels of oligomerization, Eur. J. Biochem. 221 (1994) 327^334. R. Benndorf, B. Haye, S. Ryanzantsev, M. Wieske, J. Behlke, G. Lutsch, Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity, J. Biol. Chem. 269 (1994) 20780^20784.
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A. Beall et al. / Biochimica et Biophysica Acta 1449 (1999) 41^49 [41] A. Zantema, M. DeVries, D. Maasdam, S. Bol, A. van der Eb, Heat shock protein 27 and aB-crystallin can form a complex, which dissociated by heat shock, J. Biol. Chem. 267 (1992) 12936^12941. [42] K. Kato, K. Hasegawa, S. Goto, Y. Inaguma, Dissociation as a result of phosphorylation of an aggregated form of the small stress protein HSP27, J. Biol. Chem. 269 (1994) 11274^ 11278.
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[43] A. Shiraz, K. Iizuka, P. Fadden, C. Mosse, A.P. Somlyo, A.V. Somlyo, A.J. Haystead, Puri¢cation and characterization of the mammalian myosin light chain phosphatase holoenzyme, J. Biol. Chem. 269 (1994) 31598^31606. [44] M. Gaestel, R. Benndorf, K. Hayess, I. Priemer, K. Engel, Dephosphorylation of the small heat shock protein, HSP25, by calcium/calmodulin-dependent (type 2B) protein phosphatase, J. Biol. Chem. 267 (1992) 21607^21611.
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Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of speci¢c contractile regulatory proteins Arthur Beall
b;d
, Aaron Epstein
a;d
, David Woodrum
c;d
, Colleen M. Brophy
a;b;c;d;
*
a
b
Department of Surgery, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA Department of Medicine (Institute for Molecular Medicine and Genetics), Medical College of Georgia, Augusta, GA 30912, USA c Department of Cell Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA d Augusta Veterans Administration Medical Center, Augusta, GA 30901 USA Received 17 November 1998; accepted 23 November 1998
Abstract Cyclosporine A (CSA) is a type 2B phosphatase inhibitor which can induce contraction of renal artery smooth muscle. In this investigation, we examined the phosphorylation events associated with CSA-induced contraction of bovine renal artery smooth muscle. Contractile responses were determined in a muscle bath and the corresponding phosphorylation events were determined with whole cell phosphorylation and two-dimensional gel electrophoresis. CSA-induced contractions were associated with increases in the phosphorylation of the 20 kDa myosin light chains (MLC20 ) and different isoforms of the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions was associated with increases in the phosphorylation of another small heat shock protein, HSP20, and decreases in the phosphorylation of the MLC20 , and some isoforms of HSP27. These data suggest that CSA-induced contraction and relaxation of vascular smooth muscle is associated with increases in the phosphorylation of specific contractile regulatory proteins. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Cyclosporine; Smooth muscle, vascular; Myosin light chain; Heat shock protein
1. Introduction Cyclosporine A (CSA) is an immunosuppressant drug used for the prevention of graft rejection after transplantation. The clinical usefulness of CSA is limited by vascular toxicity, which is manifest by systemic hypertension and nephrotoxicity [1,2]. CSA therapy leads to an increase in renal vascular
* Corresponding author, at address a. Fax: (706) 8233949; E-mail: [email protected]
resistance and a decrease in renal blood £ow in humans and in animal models [3,4]. It has been proposed that CSA decreases renal blood £ow by inducing the release of vasoconstrictor substances, decreasing the endothelial production of vasorelaxants, or by a direct e¡ect on the vascular smooth muscle [5^8]. The e¡ects of CSA on the vascular smooth muscle include enhanced responsiveness to vasoconstrictors, decreased responsiveness to vasorelaxants, and a direct contractile e¡ect on rat aortic and bovine renal smooth muscle [6,8^11]. The proposed mechanism of action of CSA as an
0167-4889 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 9 8 ) 0 0 1 6 9 - 4
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immunosuppressant is via binding to the intracellular cyclophilins and inactivating the Ca2 - and calmodulin-dependent protein phosphatase (type 2B phosphatase otherwise known as calcineurin) in T-lymphocytes [12]. Since other phosphatase inhibitors, such as okadaic acid and calyculin, are potent mediators of vascular smooth muscle contraction [13,14], we hypothesized that CSA-induced vascular smooth muscle contractions would be associated with increases in the phosphorylation of speci¢c contractile regulatory proteins. 2. Materials and methods 2.1. Materials CSA was from Sandoz (East Hanover, NJ); [ P]orthophosphate from Amersham (Arlington Heights, IL); N-2-hydroxyethylpiperazine-NP-2-ethanesulfonic acid (HEPES), ethylene glycol-bis(L-amino ethyl ether) N,N,NP,NP-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), cremophore, and all other analytical grade chemicals were from Sigma (St. Louis, MO). Mouse monoclonal anti-HSP27 antibodies were from Dr. M.J. Welsh (University of Michigan, Ann Arbor, MI) [15] and rabbit polyclonal anti-MLC20 antibodies were from Dr. J. Stull (University of Texas, Dallas, TX). Goat anti-mouse secondary antibodies were from Jackson Immunochemical (West Grove, PA). Electrophoresis reagents and the DC protein assay kit were from Bio-Rad (Hercules, CA). 32
2.2. Muscle bath experiments Bovine renal arteries were obtained from an abattoir and placed directly in cold HEPES bu¡er (140 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4 , 1.0 mM NaH2 PO4 , 1.5 mM CaCl2 , 10 mM glucose, and 10 mM HEPES, pH 7.4). The vessels were opened longitudinally and the adventitia was dissected free of the vessel. The endothelium was removed by rubbing the strip with a cotton tipped applicator. Transverse strips, 1.0 mM in diameter, were cut, and a loop of 3-0 silk was tied to each end. The strips were suspended in a muscle bath containing bicarbonate bu¡-
er (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4 , 1.0 mM NaH2 PO4 , 10 mM glucose, 1.5 mM CaCl2 , and 25 mM Na2 HCO3 , pH 7.4), equilibrated with 95% O2 /5% CO2 , at 37³C for at least 1 h. Changes in isometric force were registered by tension transducers (Grass, Quincy, MA) and recorded on a chart recorder (Gould, Norcross, GA). The strips were progressively stretched and the isometric force generated in response to 110 mM KCl (made with equimolar replacement of NaCl in bicarbonate bu¡er) was determined until optimal tension was produced. CSA and forskolin were solubilized in DMSO and then added directly to the muscle bath at dilutions greater than 1/100. DMSO, 1/100 dilution, had no e¡ect on smooth muscle contractile responses (data not shown). 2.3. Whole cell phosphorylation and two-dimensional gel electrophoresis Strips of renal artery smooth muscle were equilibrated in bicarbonate bu¡er bubbled with 95% O2 / 5% CO2 for 1 h at 37³C. The strips were then rinsed and incubated in low-phosphate bu¡er (10 mM HEPES pH 7.4, 140 mM NaCl, 4.7 mM KCl, 1.0 mM MgCl2 , 1.5 mM CaCl2 , 10 mM glucose, and 0.3 mM NaH2 PO4 ) for 30 min. The strips were then equilibrated in low-phosphate bu¡er containing 250 WCi/ml [32 P]orthophosphate for 1 h. The strips were treated with CSA (0.4 mM for 10 min) or remained in bu¡er as controls. The doses of CSA used in this study were the half-maximal dose of CSA required for contraction of bovine renal artery smooth muscle in vitro [11]. The incubation was terminated by immersing the muscle strips in a dry iceacetone slurry and then crushing the tissue with mortar and pestle under liquid N2 . The powder was resuspended in homogenization bu¡er (20 mM HEPES pH 7.4, 20 mM sucrose, 100 mM NaF, 15 mM EDTA, 2 mM EGTA, 1 mM PMSF, and 1.3% SDS) and boiled for 5 min. Two-dimensional gel electrophoresis was performed using vertical slab isoelectric focusing gels with the modi¢cation described by Hochstrasser et al. [16]. The conditions were optimized to emphasize the low molecular weight phosphoproteins (in the range of MLC20 ). Brie£y, the samples were acetone-precipitated and reconstituted in 9 M urea and
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2% CHAPS. The protein concentrations of the samples were determined and 150 Wg of protein were adjusted to a ¢nal concentration of 9 M urea, 2% CHAPS, 100 mM dithiothreitol (DTT), 15% glycerol, and 5% ampholines (5 parts 6^8, 3 parts 5^7, 2 parts 3^10). The ¢rst dimensions were focused for 10 000 Vh, and then run on a 12% SDS-PAGE second dimension [17]. The gels were stained with Coomassie brilliant blue, dried, and exposed to Kodak XAR ¢lm. For immunoblotting, second dimension PAGE gels were transferred to Immobilon-P (Millipore, Bedford, MA), blocked with TBS-milk (10 mM Tris (pH 7.4), 150 mM NaCl, 5% milk), washed with TBS-Tween (10 mM Tris (pH 7.4), 150 mM NaCl, 0.5% Tween 20), and probed with mouse anti-HSP27 antibody (1:5000 in TBS-milk for 1 h). Immunoreactive proteins were detected using peroxidase conjugated goat anti-mouse (1:2000 in block bu¡er) and Western blot chemiluminescence reagent (DuPont-NEN, Boston, MA).
43
non-phosphorylated MLC20 were quantitated with a PhosphorImager. 2.5. Statistical analysis Values are reported as mean þ S.E.M. and n refers to the number of animals examined. The statistical di¡erences between groups were determined with one way repeated measures analysis of variance (ANOVA) using Sigma Stat software (Jandel Scienti¢c, San Rafael, CA). A P value less than 0.05 was considered signi¢cant. Densitometric analysis of immunoreactive HSP27 was performed using a CCD camera interfaced with a 2D analyzer software package (Innovision Software, Bioimage, Ann Arbor, MI). The MLC20 bands and phosphorylated proteins on two-dimensional gels were quantitated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software (Molecular Dynamics).
2.4. MLC20 phosphorylation Strips of bovine renal artery smooth muscle were equilibrated in a muscle bath as described above. The strips were kept in bu¡er (control) or stimulated with KCl (110 mM) or CSA (0.4 mM). When a maximal contractile response was obtained (after 10 min), the muscles were immediately snap frozen with liquid N2 cooled tongs and ground to a ¢ne powder under liquid N2 . The powder was placed in 90% acetone, 10% trichloroacetic acid, 10 mM DTT and subsequently washed 3 times with 100% acetone, 10 mM DTT. The pellet was resuspended in 9 M urea, 2% CHAPS, 100 mM DTT and protein concentrations determined. Twenty Wg of protein was separated on glycerol-urea mini-gels (40% glycerol, 15% acrylamide, 0.75% bisacrylamide, 10 mM Tris, and 22 mM glycine) at 150 V for 19 h in the cold room [18]. The proteins were then transferred to Immobilon (Millipore, Bedford, MA), 100 V for 1 h. The blots were blocked with TBS-milk and then incubated overnight with anti-MLC20 antibodies (1:4000 in TBS-milk). After washing (6 times with TBS-Tween), the blots were incubated in 125 I-protein A and the relative amounts of phosphorylated and
Fig. 1. CSA-induced contractions of renal artery smooth muscle. Strips of renal artery smooth muscle were equilibrated in a muscle bath and after the initial response to high extracellular KCl (110 mM) was determined, the strips were contracted with CSA (0.4 mM). The CSA was then washed out (W, panel A), or remained in the bath and sodium nitroprusside (10 WM, S, panel B), or forskolin (10 WM, F, panel C) was added.
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centrations (1500^2000 ng/ml) that can occur in clinical settings.
3. Results 3.1. CSA is a direct vascular smooth muscle contractile agonist CSA (0.4 mM) induces contraction in bovine renal artery smooth muscles which relax when the CSA was washed out (100% relaxation, n = 6, Fig. 1); or with the addition of the vasorelaxants, sodium nitroprusside (10 WM, 94 þ 3% relaxation, n = 8, Fig. 1); or forskolin (10 WM, 88 þ 6% relaxation, n = 6, Fig. 1). The dose of CSA used in this study represents the dose necessary for half-maximal contraction of bovine renal artery smooth muscle [11]. This dose is higher than optimal serum levels of CSA (250^350 ng/ml); however, it approaches the peak serum con-
3.2. Phosphorylation events associated with CSA-induced contractions in renal artery smooth muscle Whole cell phosphorylation and two-dimensional gel electrophoresis of strips of renal artery smooth muscles treated with CSA (0.4 mM for 10 min) led to increases in the phosphorylation of speci¢c phosphoproteins. There were increases in the phosphorylation of multiple 27 kDa proteins with di¡erent isoelectric points (Fig. 2, Table 1). Bitar and coworkers have suggested that phosphorylation of the 27 kDa small heat shock protein, HSP27, is involved in sustained
Fig. 2. CSA-induced phosphorylation changes in bovine renal artery smooth muscle. Strips of bovine renal artery smooth muscle were loaded with [32 P]orthophosphate and equilibrated in bu¡er (panels A, D, G), treated with cyclosporine A (CSA, 0.4 mM, for 10 min, panels B, E, H) or treated with cyclosporine A (0.4 mM for 10 min) followed by forskolin (10 WM for 10 min, panels C, F, I). The strips were homogenized and the proteins separated by isoelectric focusing in the ¢rst dimension (indicated by pI 4.5^6.5) and SDSPAGE in the second (the relative mobility of the proteins is indicated by the molecular weight markers on the left of the gels). The proteins were transferred to Immobilon and the membrane was developed with a PhosphorImager (autoradiographs are depicted in panels A, B, C). The membranes were then probed with anti-HSP27 antibodies (panels D, E, F) and re-probed with anti-HSP20 antibodies (panels G, H, I). The location of the myosin light chains (M), the isoforms of HSP27 (A, B, C), and the isoforms of HSP20 (Nos. 8, 3, 4, as previously labeled by Takuwa [22]) are indicated on the blots. Non-phosphorylated, immunoreactive HSP20 and HSP27 are also indicated. These blots are representative of ¢ve separate experiments.
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Table 1 Quantitation of phosphorylation changes induced by CSA alone and CSA followed by forskolin in bovine renal artery smooth muscle Protein
CSA
CSA/FSK
Reference MLC20 HSP27A HSP27B HSP27C HSP20 #4 HSP20 #3 HSP20 #8
1.07 þ 0.08 2.14 þ 0.65* 1.76 þ 0.26* 1.78 þ 0.19* 1.83 þ 0.21* 2.01 þ 0.14* 1.35 þ 0.21 2.06 þ 0.90*
1.02 þ 0.13 0.72 þ 0.27 1.32 þ 0.26 1.29 þ 0.22 1.76 þ 0.51* 2.26 þ 0.53* 2.40 þ 0.33* 5.40 þ 0.60*
The values are the mean of the fold increase over control þ S.E.M. for cyclosporine (CSA, 0.4 mM, 10 min) and CSA (0.4 mM, 10 min) followed by forskolin (FSK, 10 WM, 10 min) treated strips (n = 5, *P 6 0.05, ANOVA compared to control). `Reference' refers to the same reference protein identi¢ed by Takuwa et al. [22]; HSP27A is the isoform of HSP27 with a pI of 5.5; HSP27B, pI 5.7; HSP27C, pI 5.9; HSP20 #4 is the isoform of HSP20 with a pI of 6.0; HSP20 #3, pI 5.9; and HSP20 #8, pI 5.7.
smooth muscle contraction [18]. To determine whether the 27 kDa proteins, which demonstrated an increase in phosphorylation after CSA treatment were HSP27, two-dimensional gels were transferred
Fig. 4. CSA treatment leads to increases in the phosphorylation of MLC20 . Strips of bovine renal artery smooth muscle were equilibrated in a muscle bath and after an initial high KCl contraction, remained in bu¡er (CONT), or were treated with high KCl (110 mM, for 5 min), or CSA (0.4 mM, 10 min). The samples were separated on glycerol-urea gels [18], and probed with antibodies against MLC20 . A representative blot is depicted in panel A and the arrow identi¢es the phosphorylated isoform of MLC20 . Densitometric analysis of multiple blots is depicted in panel B (n = 5 di¡erent strips, error bars depict standard error of the mean, *P 6 0.05, ANOVA).
Fig. 3. CSA treatment leads to increases in the immunoreactive isoform of HSP27 in bovine renal artery smooth muscle. Strips of bovine renal artery smooth muscle were equilibrated as controls (CONT) or treated with CSA (0.4 mM for 10 min) and the proteins were separated on two-dimensional gels. The proteins were transferred to Immobilon and probed with antiHSP27 antibodies. The relative density of the spots was quantitated as described in the speci¢c methods and the fraction of the immunoreactive acidic isoform of HSP27 (isoform A) expressed as a percentage of the sum of the total immunoreactive HSP27 (n = 5, error bars indicate standard error of the mean, *P 6 0.05 ANOVA).
to Immobilon and probed with anti-HSP27 antibodies. Proteins immunoreactive with anti-HSP27 antibodies demonstrated a similar mobility on two-dimensional gels as the 27 kDa phosphoproteins (Fig. 2). There was an immunoreactive spot with a relative mobility of 27 kDa which did not correspond to a phosphorylated 27 kDa protein. This likely represents non-phosphorylated HSP27. Densitometric analysis of the immunoblots demonstrated a signi¢cant increase in the amount of the most acidic isoform (isoform A) of HSP27 as a fraction of the total immunoreactive HSP27 after CSA treatment (Fig. 3). CSA treatment (0.4 mM for 10 min) also led to increases in the phosphorylation of the MLC20 . Proteins from muscles treated with or without CSA (0.4 mM, for 10 min) were separated on glycerol-urea gels and probed with antibodies against MLC20 [19]. Proteins from muscles treated with high KCl were used as positive controls. Treatment with CSA led to increases in the phosphorylation of the MLC20 that
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were similar to the increases that occurred with high extracellular KCl (Fig. 4). Forskolin-induced relaxation of renal artery smooth muscles that had been pre-contracted with CSA led to decreases in the phosphorylation of the MLC20 some isoforms of HSP27 (Table 1). Cyclic nucleotide-dependent relaxation was associated with increases in the phosphorylation of three 20 kDa proteins (Table 1). These proteins have recently been identi¢ed as di¡erent isoforms of the small heat shock protein, HSP20 [20]. Proteins immunoreactive with anti-HSP20 antibodies demonstrated a similar mobility on two-dimensional gels as the 20 kDa phosphoproteins (Fig. 2). 4. Discussion Chronic administration of CSA signi¢cantly and reproducibly produces hypertension in humans and animal models [1]. This may be due to a direct e¡ect of CSA on the vascular smooth muscle [8,11]. Increases in the phosphorylation of a number of proteins including the myosin light chains (MLC20 ), desmin, caldesmon, and the small heat shock protein, HSP27, have been associated with the contractile state of vascular smooth muscles [21^23]. Increases in the phosphorylation of these proteins may occur with activation of speci¢c kinases which are capable of phosphorylating the speci¢c proteins, or inhibition of the phosphatases which de-phosphorylate these proteins [24]. CSA is a speci¢c inhibitor of Ca2 / calmodulin-dependent phosphatase (phosphatase 2B or calcineurin) [12]. Inhibitors of type 1 and 2A phosphatases have been shown to directly induce vascular smooth muscle contraction, presumably as a result of an inhibition of the de-phosphorylation of speci¢c proteins [13,14]. However, the e¡ect of CSA, a type 2B phosphatase inhibitor, on protein phosphorylation events in vascular smooth muscles has not been previously examined. In this investigation we determined that CSA-induced contractions are associated with increases in the phosphorylation of a set of speci¢c proteins similar to those that become phosphorylated during agonist-induced contractions in multiple types of smooth muscles [21^23]. Stimulation of renal artery smooth muscle with CSA led to increases in the
phosphorylation of MLC20 (Table 1, Fig. 4). Increases in the phosphorylation of the MLC20 are thought to regulate the initiation of smooth muscle contraction [25]. However, sustained vascular smooth muscle contraction has been described in many systems in which the state of MLC20 phosphorylation returns to its baseline value during the sustained phase of contraction [26]. Thus, numerous investigators have implicated other phosphorylation events in mediating the sustained phase of contraction [27^29]. CSA treatment also led to increases in the phosphorylation of the small heat shock protein, HSP27 (Table 1, Figs. 2 and 3). Other investigators have noted increases in the phosphorylation of 27 kDa proteins that is temporally associated with smooth muscle contraction [23]. Increases in the phosphorylation of HSP27 is also associated with carbachol stimulated tracheal smooth muscle contraction [30] and thrombin-induced vascular smooth muscle contractions [31]. Prolonged treatment of animals with CSA leads to an attenuation of the endothelial-independent relaxation response to sodium nitroprusside [10]. In addition, vascular smooth muscle contractions induced by the type I/IIa phosphatase inhibitor, calyculin, are refractory to relaxation with the addition of sodium nitroprusside [32]. However, contractions induced by the acute administration of CSA in a muscle bath relax rapidly with the addition of the guanylate cyclase activator, sodium nitroprusside or the adenylate cyclase activator, forskolin (Fig. 1). Cyclic nucleotide-dependent relaxation of CSA-precontracted renal artery smooth muscles is associated with decreases in the phosphorylation of the MLC20 and some isoforms of HSP27 (Table 1). Relaxation was also associated with increases in the phosphorylation of two 20 kDa proteins that have recently been identi¢ed as HSP20 (Fig. 2, Table 1) [20]. Heat shock proteins function as molecular chaperones which confer resistance to cellular stresses. While the function of HSPs has been well established in cultured cells, less is known about the actual physiologic functions of HSPs in intact tissues. These data and the data of others suggest that the small HSPs may play a role in modulating the contractile state of smooth muscles. Bitar and co-workers have recently implicated HSP27 as a mediator of sustained
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rectal sphincter smooth muscle tone [18]. HSP27 has been shown to be relatively abundant in muscle tissue [33] and recent data suggest that HSP27 interacts with and regulates the actin cytoskeleton [34^37]. HSP20 was initially identi¢ed as a byproduct of the puri¢cation of HSP27 and increases in the phosphorylation of HSP20 are associated with cyclic nucleotide-dependent relaxation of smooth muscle [20,38]. The small HSPs exist intracellularly as large macromolecular aggregates [39^42]. Thus, it is possible that the oligomeric association of HSP27 and HSP20 functions as a molecular chaperone to stabilize the contractile machinery resulting in sustained contraction and that phosphorylation of HSP20 disrupts this interaction leading to relaxation. Several phosphatases have been shown to use phosphorylated MLC20 as a substrate in vitro and the majority of these phosphatases have been classi¢ed as type 1 or 2A. A myosin bound phosphatase has been identi¢ed that is a trimeric type 1 enzyme [43]. Increases in the phosphorylation of MLC20 in vascular smooth muscle treated with CSA suggest that type 2B phosphatase may also regulate MLC20 phosphorylation. The Ca2 /calmodulin-dependent protein phosphatase (calcineurin) has been implicated in the de-phosphorylation of HSP25, the murine analogue of bovine and human HSP27 [44]. Since the activation of many kinases is dependent on phosphorylation, it is also possible that the increases in phosphorylation of speci¢c smooth muscle proteins associated with CSA treatment are due to an inhibition of the de-phosphorylation of speci¢c kinases leading to persistent activation of the kinases. In summary, CSA-induced renal artery smooth muscle contractions are associated with increases in the phosphorylation of a similar speci¢c set of proteins as occurs with agonist-induced contraction. One of these proteins was identi¢ed as the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions are associated with decreases in the phosphorylation of these regulatory proteins and with increases in the phosphorylation of the small heat shock protein, HSP20. CSA may directly induce vascular smooth muscle contraction by speci¢cally inhibiting the de-phosphorylation of smooth muscle regulatory proteins or by inhibiting the de-phosphorylation of kinases thus producing persistent activation of the kinases.
47
Acknowledgements This work was supported by a grant from the VA Merit Review, the NIH (1RO1HL58027-02), and an American Heart Association Clinician Scientist Award (to Dr. C.M. Brophy). The authors appreciate the assistance of Shapiro's Meatpacking Corp. for donating bovine blood vessels. The authors also appreciate the donation of anti-MLC20 antibodies from Dr. James Stull and anti-HSP27 antibodies from Dr. Michael Welsh. References [1] D.V. Hamilton, D.J.S. Carmichael, D.B. Evans, R.Y. Calne, Hypertension in renal transplant recipients on cyclosporine A and corticosteroids and azathioprine, Transplant. Proc. 14 (1982) 597^600. [2] B.D. Myers, J. Ross, L. Newton, L. Luetscher, M. Perlroth, Cyclosporine-associated chronic nephropathy, New Engl. J. Med. 311 (1984) 699^705. [3] J. English, A. Evan, D.C. Houghton, W.M. Bennett, Cyclosporine-induced renal dysfunction in the rat, Transplantation 44 (1987) 135^141. [4] M.R. Weir, D.K. Klassen, S.Y. Shen, D. Sullivan, E.U. Buddenmeyer, B.S. Handwerger, Acute e¡ects of intravenous cyclosporine on blood pressure, renal hemodynamics, and urine prostaglandin production of healthy humans, Transplantation 49 (1990) 41^47. [5] D.M. Lanese, J.D. Conger, E¡ects of endothelin receptor antagonist on cyclosporine-induced vasoconstriction in isolated rat renal arterioles, J. Clin. Invest. 91 (1993) 2144^ 2149. [6] A. Rego, R. Vargas, M.L. Foegh, P.W. Ramwell, E¡ect of cyclosporine A treatment on vascular reactivity of the rat thoracic aorta, Transplant. Proc. 20 (1988) 572^577. [7] K. Sudir, J.S. MacGregor, T. DeMarco, C.J.M. DeGroot, R.N. Taylor, T.M. Chou, P.G. Yock, K. Chatterjee, Cyclosporine impairs release of endothelium-derived relaxing factors in epicardial and resistance coronary arteries, Circulation 90 (1994) 3018^3023. [8] H. Xue, R.D. Bukowki, D.A. McCarron, W.M. Bennet, Induction of contraction in isolated rat aorta by cyclosporine, Transplantation 43 (1987) 715^718. [9] M.D. Gar, M.S. Paller, Cyclosporine augments renal but not systemic vascular reactivity, Am. J. Physiol. 258 (1990) F211^F217. [10] H.C. Huang, A. Rego, R. Vargas, M.D. Foegh, P.W. Ramwell, Nitroprusside-induced vascular relaxation is attenuated in organ-transplanted animals treated with cyclosporine, Transplant. Proc. 19 (1987) 126^130. [11] A. Epstein, J. Wynn, L. Mulloy, C.M. Brophy, Cyclosporine, but not FK506, selectively induces renal and coronary
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