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Glucocorticoids Inhibit the Expression of Calcium-Dependent Potassium Channels in Vascular Smooth Muscle
Molecular Genetics and Metabolism 67, 53–57 (1999) Article ID mgme.1999.2812, available online at http://www.idealibrary.com on
Glucocorticoids Inhibit the Expression of Calcium-Dependent Potassium Channels in Vascular Smooth Muscle Andrew S. Brem, 1 Robert B. Bina, Sunil Mehta, and John Marshall Brown University, Departments of Pediatrics (Nephrology) and Biophysics, Providence, Rhode Island 02903 Received November 17, 1998, and in revised form January 25, 1999
Key Words: hypertension; glucocorticoids; vascular smooth muscle; 11b-hydroxysteroid dehydrogenase; carbenoxolone; calcium-dependent potassium channels.
Increased calcium-activated potassium channel (K Ca) activity in vascular smooth muscle (VSM) cells leads to a relaxation response counteracting the effects of high blood pressure. Since chronic exposure to glucocorticoids (GC) can be associated with an increase in blood pressure, we reasoned that GCs might modify the expression of K Ca channels resulting in a net rise in vascular tone. To test this hypothesis, primary cultures of rat VSM cells were exposed to (a) RU 28362 (a pure glucocorticoid receptor agonist), 1 mM; (b) corticosterone 10 nM 1 carbenoxolone (an inhibitor of bidirectional VSM 11b-OH steroid dehydrogenase), 1 mM; (c) 11-dehydrocorticosterone (a biologically inactive metabolite), 10 nM 1 carbenoxolone; (d) carbenoxolone alone; or (e) aldosterone 10 nM for periods of up to 72 h. Proteins were then extracted and Western blots prepared. Gels were probed with a rabbit-derived polyclonal antibody directed against K Ca channel protein. The experimental procedure was repeated on separate sets of VSM cells to ensure reproducibility. Expression of K Ca channel protein was diminished in VSM cells incubated with corticosterone 1 carbenoxolone and with RU 28362 after 24 h and remained low at 72 h. Expression of K Ca protein in cells exposed to 11-dehydrocorticosterone 1 carbenoxolone, carbenoxolone alone, and aldosterone was either similar to controls or mildly increased over the 72 h. These data are consistent with the hypothesis that GCs diminish the expression of K Ca protein. Diminished K Ca expression could contribute to the observed increase in vascular tone following chronic GC exposure. © 1999 Academic Press
Blood pressure is regulated by a complex series of physical and hormonal processes. Glucocorticoids have been shown to indirectly or directly alter many of these processes including renal sodium balance and vascular tone (2,5,8,13,14,16,18,24). How glucocorticoids affect the vascular tree has been a subject of intense interest to many investigators given the association of glucocorticoids with hypertension and the widespread use of these steroids in clinical medicine. In humans, both cortisol and synthetic glucocorticoids like dexamethasone increase the sensitivity to applied pressors (18,23). Experiments conducted in various animal and cellular models have confirmed the observation that glucocorticoids have the ability to enhance the contractile response to pressors (4,6,11,12). We have observed that the contractile response of rat aortic rings is enhanced if those rings are incubated with physiologic concentrations of corticosterone and an inhibitor of the enzyme 11b-hydroxysteroid dehydrogenase in vascular smooth muscle which is responsible for local glucocorticoid metabolism (4,6). Moreover, glucocorticoids have been linked to increased hepatic generation of angiotensinogen (9), an increase in angiotensin II type I receptors (21), an upregulation of V1a vasopressin and alpha adrenergic receptors in vascular smooth muscle (15,20), and a decrease in basal guanylate cyclase activity in vascular tissue (25). Calcium-dependent potassium channels con-
1
To whom correspondence should be addressed at Division of Pediatric Nephrology, Rhode Island Hospital, APC 942, 593 Eddy Street, Providence, RI 02903. Fax: (401) 444-3944. E-mail: [email protected]. 53
1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
54
BREM ET AL.
tained in the membrane of vascular smooth muscle cells have recently been shown to play a role in attenuating the vasoconstrictor effects seen in various forms of hypertension (19). When activated, these channels allow for the efflux of potassium from the cell resulting in hyperpolarization and relaxation. To date, there is little in the literature on hormonal control over the expression or activity of this channel. Given the net increase in vascular tone with chronic exposure to glucocorticoids, we hypothesized that these steroids may alter the expression of this form of potassium channel in vascular smooth muscle. In the following studies, we demonstrate that glucocorticoids but not mineralocorticoids have the ability to decrease the expression of potassium channels. METHODS Primary Vascular Smooth Muscle (VSM) Cell Cultures The aortas from eight sacrificed adult SpragueDawley rats were dissected under sterile conditions and associated fat and connective tissue removed. The aortas were next incubated at 37°C for 15 min with digestion mixture which consists of collagenase (1 mg/ml), elastase (0.125 mg/ml), DMEM/F12 (Gibco BRL, Grand Isle, NY) with Hepes (pH 7.4), penicillin (100 mg/ml), and streptomycin (100 mg/ ml). At the completion of the incubation, the adventitia was carefully stripped, the vessel longitudinally opened, and the luminal surface gently scraped with forceps to remove endothelial cells. The remaining tissue was then minced into 1-mm pieces and incubated with digestion mixture for an additional 90 min at 37°C using a shaking water bath. The digestion was stopped with 8 ml of DMEM/F12, 10% fetal calf serum, and 25 mM Hepes. Following digestion, the remnant tissue became a suspension of smooth muscle cells. The cells were passed over a sieve and centrifuged at 1000 rpm for 5 min. Supernatant was discarded and the cells resuspended in DMEM/F12, 10% fetal calf serum, and 25 mM Hepes. The cells were then plated at a concentration of 1 3 10 4 viable cells/cm onto T-25 plates and placed in an incubator. The flasks were gassed with 5% CO 2-95% O 2 and were maintained at 37°C. Cells studied up to 40 passages have shown to retain the expression of a-actin (7), a factor unique to vascular smooth muscle cells (27). Cell counts were obtained before each plating and
prior to any experiment. The medium for the cells was changed twice per week. Studies were usually conducted on the cells after 5 to 7 days when the cells have reached confluence and when they are in a quiescent stage. Cells were made quiescent by removing all growth factors including fetal serum from the incubation medium for 24 h prior to exposure to steroids. Western Blots Vascular smooth muscle cells were grown to confluence in 6-well plates (Corning-Costar, Cambridge, MA) with DMEM/F12 (Gibco BRL) and 10% fetal bovine serum (Sigma Chemical). After reaching confluence, the cells were transferred to serum-free DMEM/F12 for 24 h. At 24 h, the various test compounds were added to the flasks and the incubations were continued for 1 to 3 days. The cells were then rinsed with serum-free DMEM/F12 and scraped off with a cell scraper and 100 mL of running buffer for each well. Protein concentrations were determined with the Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Richmond, CA). Eppendorf tubes containing the samples were boiled for approximately 7 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 8% polyacrylamide gels. Equal amounts of protein were loaded at 100 mg per lane and electrophoretically blotted onto Immobilon-P transfer membranes (Millipore, Bedford MA). The Immobilon membranes were then incubated in TBST (TRIS-buffered saline with 0.05% Tween 20) containing 5% nonfat dry milk (wt/vol) for 30 min at room temperature on a rocking platform, followed by overnight incubation at 4°C in the desired antibody (Bslo K 1 channel rabbit polyclonal antibody) at a 1:250 dilution in fresh TBST-5% nonfat milk buffer. This potassium channel antibody was kindly provided by Dr. Edward Moczydlowski, Department of Pharmacology and Cellular and Molecular Physiology, Yale University School of Medicine. After incubation, the membranes were washed three times for 7 min each in 1X TBST and incubated with HRP-conjugated complementary antibody (Anti Rabbit IgG (whole molecule) peroxidase conjugate, Sigma Chemical, Cat No. A-6154; 1:1000 dilution) in blocking buffer (TBST with 5% nonfat dry milk) for 30 min. The membranes were then washed four times with 1X TBST and exposed to ECL reagent (Amersham Life Science, Buckinghamshire, England) for 1–3 min. The membranes were semidried between two sheets
GLUCOCORTICOIDS INHIBIT K 1 CHANNELS
FIG. 1. A representative Western blot showing the expression of the calcium-dependent potassium channel protein at Day 0 (1 h) and Day 1. For purposes of comparison, the expression of the potassium channel protein from control cells, cells incubated with carbenoxolone (CBX), 1 mM, and cells incubated with the glucocorticoid agonist RU 28362 (RU 362), 1 mM, are shown. Expression of the potassium channel protein was decreased in vascular smooth muscle cells incubated for 24 h with the glucocorticoid RU 28362.
of filter paper and quickly exposed to ECL Hyperfilm (Amersham Life Sciences) for 20 min to 1 h before being developed. The results were analyzed by densitometric scanning using a Power Macintosh running NIH Image 1.60 and expressed as relative increase compared to controls. Where appropriate, results are presented as the density of the experimental sample relative to the density of controls run at the same time under identical conditions. Where appropriate, data were analyzed by Student’s t test with P values less than 0.05 considered significant.
55
hydrogenase inhibitor carbenoxolone alone (1 mM), aldosterone (10 nM), the inactive metabolite, 11dehydrocorticosterone (10 nM) plus carbenoxolone, or the mineralocorticoid antagonist RU 318 (1 mM). From 24 to 72 h, cells exposed to the pure glucocorticoid RU 28362 and cells exposed to corticosterone (10 nM) plus the licorice derivative carbenoxolone (1 mM), which prevents the metabolism of the corticosterone, showed a decrease in the expression of this potassium channel (Fig. 2). The 25 to 30% decrease in potassium channel expression observed with RU 28362 and with corticosterone plus carbenoxolone was not significantly different between 24 and 72 h. As mentioned, vascular smooth muscle cells incubated with aldosterone (10 nM) also showed no clear effect on the potassium channel protein but the expression was considerably more varied. DISCUSSION Glucocorticoid receptors are present in aorta, in mesenteric arteries, and in cultured vascular smooth muscle cells (10). These steroids can bind to and activate these receptors resulting in enhanced vasoconstriction following stimulation with cat-
RESULTS Primary cultures of rat vascular smooth muscle cells were incubated with vehicle or vehicle containing the test reagent(s) for 1, 24, and for 72 h. When the blots at each time point were scanned and the relative density of each blot analyzed compared to its own control, no differences in the expression of the calcium-dependent potassium channel protein were observed at 1 h. As can be seen in a sample Western blot at 24 h, cells exposed to RU 28362 (1 mM) demonstrated a decrease in the expression of the calcium-dependent potassium channel protein (Fig. 1). There was no clear change in the total protein expression relative to control observed in cells previously exposed to the 11b-OH steroid de-
FIG. 2. Scanned densities of the potassium channel Western blots relative to controls. Results are expressed as mean experimental/control 6 SE. Values less than 1 indicate a decreased expression of the potassium channel protein. Since there was no additional change in protein expression after 24 h, the results at 24 h were combined with those at 72 h. Vascular smooth muscle cells appeared to consistently express lower amounts of the potassium channel protein after exposure to RU 28362 [RU362](1 mM) or corticosterone [B](10 nM) plus carbenoxolone [CBX](1 mM). Cells incubated with the inactive metabolite of corticosterone, 11-dehydrocorticosterone [A], plus carbenoxolone did not demonstrate a decreased expression of the potassium channel.
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echolamines and angiotensin II (18,23). The enhanced constrictor response following chronic glucocorticoid exposure is related, in part, to a documented increased influx of sodium and calcium ions into the vascular smooth muscle cell (12). These changes in intracellular ion concentration may be explained, in part, by a glucocorticoid-induced decrease in the sodium-calcium ion exchanger (22). All these glucocorticoid-induced alterations in membrane permeability fit nicely with the rapidly evolving hypotheses linking changes in intracellular ion concentrations and transport pathways to various forms of hypertension (1,3). Vascular smooth muscle cells also contain several types of potassium channels including the calcium-activated or -dependent potassium channel (17). Activation of this channel leads to an efflux of potassium from the cell, a hyperpolarized transmembrane potential, and vasodilator response. The calcium-dependent potassium channel is activated with various forms of hypertension in an effort to blunt the rise in blood pressure. Thus far, the arachidonic acid derivative 11,12 EET (26) and physical forces like stretch have been shown to directly activate this channel in vascular smooth muscle (17). The current preliminary observations provide evidence that glucocorticoids directly affect the expression of the calcium activated potassium ion channel in vascular smooth muscle. If the downregulated expression of this potassium channel by glucocorticoids translates into a parallel decrease in channel activity, one may expect an attenuated relaxation response and a net rise in vascular tone. The pure glucocorticoid receptor agonist RU 28362 and corticosterone plus carbenoxolone appear to selectively decrease the expression of this potassium channel protein. In contrast, carbenoxolone, an inhibitor of the bidirectional isoform of 11b-OH steroid dehydrogenase found in vascular smooth muscle (7), exerts either no effect or a mildly positive effect on the expression of the channel protein. In addition, vascular smooth muscle cells incubated with 11-dehydrocorticosterone, the inactive product of 11b-OH steroid dehydrogenase, plus carbenoxolone do not show a difference in the expression of this protein relative to controls. The mineralocorticoid aldosterone exerts no clear effect on the expression of the potassium channel protein relative to controls. However, there is a considerable scatter in the data. The steroid-induced downregulation of the potassium channel protein is not immediate; it occurs within 24 h and is maintained to at least 72 h. The
observed time line for the altered channel protein expression is consistent with glucocorticoids directly affecting a process at the nuclear level. The precise process or processes involved with this specific altered protein expression will undoubtedly require further investigation. REFERENCES 1.
Aviv A. The link between cytosolic Ca 12 and the Na 1-H 1 antiport: A unifying factor for essential hypertension. J Hypertension 6:685– 691, 1988.
2.
Baum M, Moe OW, Gentry DL, Alpern RJ. Effect of glucocorticoids on renal cortical NHE-3 and NHE-1 mRNA. Am J Physiol 267:F437–F442, 1994.
3.
Blaustein MP. Sodium ions, calcium ions, blood pressure regulation and hypertension: A reassessment and hypothesis. Am J Physiol 232:C165–C173, 1977.
4.
Brem AS, Bina RB, Hill N, Alia C, Morris DJ. Effects of licorice derivatives on vascular smooth muscle function. Life Sci 60:207–14, 1997.
5.
Brem AS, Bina RB, Ingelfinger JR. Glucocorticoids mediate angiotensin II induced electrolyte transport in immortalized renal proximal tubule cells (IRPTC). Pediatr Res 39:358A, 1996.
6.
Brem AS, Bina RB, King T, Morris DJ. 11BetaOH-progesterone affects vascular glucocorticoid metabolism and contractile response. Hypertension 30:449 –54, 1997.
7.
Brem AS, Bina RB, King T, Morris DJ. Bidirectional activity of 11 beta-hydroxysteroid dehydrogenase in vascular smooth muscle cells. Steroids 60:406 –10, 1995.
8.
Husted RF, Laplace JR, Stokes JB. Enhancement of electrogenic Na1 transport across rat inner medullary collecting duct by glucocorticoid and by mineralocorticoid hormones. J Clin Invest 86:498 –506, 1990.
9.
Klett C, Ganten D, Hellmann W, Kaling M, Ryffel GU, Weimar-Ehl T, Hackenthal E. Regulation of hepatic angiotensinogen synthesis and secretion by steroid hormones. Endocrinology 130:3660 – 8, 1992.
10.
Kornel L. Colocalization of 11 beta-hydroxysteroid dehydrogenase and mineralocorticoid receptors in cultured vascular smooth muscle cells. Am J Hypertens 7:100 –103, 1994.
11.
Kornel L, Manisundaram B, Nelson WA. Glucocorticoids regulate Na1 transport in vascular smooth muscle through the glucocorticoid receptor-mediated mechanism. Am J Hypertens 6:736 –744, 1993.
12.
Kornel L, Nelson WA, Manisundaram B, Chigurupati R, Hayashi T. Mechanism of the effects of glucocorticoids and mineralocorticoids on vascular smooth muscle contractility. Steroids 58:580 –587, 1993.
13.
Laplace JR, Husted RF, Stokes JB. Cellular responses to steroids in the enhancement of Na1 transport by rat collecting duct cells in culture. Differences between glucocorticoid and mineralocorticoid hormones. J Clin Invest 90:1370 – 1378, 1992.
14.
Lee YC, Lin HH, Tang MJ. Glucocorticoid upregulates NaK-ATPase a- and b-mRNA via an indirect mechanism in
GLUCOCORTICOIDS INHIBIT K 1 CHANNELS
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proximal tubule cell primary cultures. Am J Physiol 268: F862–F867, 1995. Murasawa S, Matsubara H, Kizima K, Maruyama K, Mori Y, Inada M. Glucocorticoids regulate V1a vasopressin receptor expression by increasing mRNA stability in vascular smooth muscle cells. Hypertension 26:665– 669, 1995. Naray-Fejes-Toth A, Fejes-Toth G. Glucocorticoid receptors mediate mineralocorticoid-like effects in cultured collecting duct cells. Am J Physiol 259:F672– 678, 1990. Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol 268:C799 –C822, 1995. Pirpiris M, Sudhir K, Yeung S, Jennings G, Whitworth JA. Pressor responsiveness in corticosteroid-induced hypertension in humans. Hypertension 19:567–574, 1992. Rusch NJ, Liu Y, Pleyte KA. Mechanisms for regulation of arterial tone by Ca21-dependent K1 channels in hypertension. Clin Exp Pharmacol Physiol 23:1077–1081, 1996. Sakaue M, Hoffman BB. Glucocorticoids induce transcription and expression of the alpha 1B adrenergic receptor gene in DTT1 MF-2 smooth muscle cells. J Clin Invest 88:385– 389, 1991. Sato A, Suzuki H, Nakazato Y, Shibata H, Inagami T, Sa-
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ruta T. Increased expression of vascular angiotensin II type 1A receptor gene in glucocorticoid-induced hypertension. J Hypertens 12:511–516, 1994. 22.
Smith L, Smith JB. Regulation of sodium-calcium exchanger by glucocorticoids and growth factors in vascular smooth muscle. J Biol Chem 269:27527–27531, 1994.
23.
Walker BR, Connacher AA, Webb DJ, Edwards CR. Glucocorticoids and blood pressure: A role for the cortisol/cortisone shuttle in the control of vascular tone in man. Clin Sci (Colch) 83:171–178, 1992.
24.
Whitworth JA. Studies on the mechanisms of glucocorticoid hypertension in humans [Review]. Blood Pressure 3:24 –32, 1994.
25.
Yasunari K, Kohno M, Murakawa K, Yokokawa K, Takeda T. Glucocorticoids and atrial natriuretic factor receptors on vascular smooth muscle. Hypertension 16:581–586, 1990.
26.
Zou AP, Fleming JT, Falck JR, Jacobs ER, Gebremedhin D, Harder DR, Roman RJ. Stereospecific effects of epoxyeicosatrienoic acids on renal vascular tone and K 1 channel activity. Am J Physiol 270:F822–F832, 1996.
27.
Clowes AW, Clowes MM, Kocher O, Ropraz P, Chaponnier C, Gabbiani G. Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin. J Cell Biol 107(5):1939 – 1945, 1988.
Glucocorticoids Inhibit the Expression of Calcium-Dependent Potassium Channels in Vascular Smooth Muscle Andrew S. Brem, 1 Robert B. Bina, Sunil Mehta, and John Marshall Brown University, Departments of Pediatrics (Nephrology) and Biophysics, Providence, Rhode Island 02903 Received November 17, 1998, and in revised form January 25, 1999
Key Words: hypertension; glucocorticoids; vascular smooth muscle; 11b-hydroxysteroid dehydrogenase; carbenoxolone; calcium-dependent potassium channels.
Increased calcium-activated potassium channel (K Ca) activity in vascular smooth muscle (VSM) cells leads to a relaxation response counteracting the effects of high blood pressure. Since chronic exposure to glucocorticoids (GC) can be associated with an increase in blood pressure, we reasoned that GCs might modify the expression of K Ca channels resulting in a net rise in vascular tone. To test this hypothesis, primary cultures of rat VSM cells were exposed to (a) RU 28362 (a pure glucocorticoid receptor agonist), 1 mM; (b) corticosterone 10 nM 1 carbenoxolone (an inhibitor of bidirectional VSM 11b-OH steroid dehydrogenase), 1 mM; (c) 11-dehydrocorticosterone (a biologically inactive metabolite), 10 nM 1 carbenoxolone; (d) carbenoxolone alone; or (e) aldosterone 10 nM for periods of up to 72 h. Proteins were then extracted and Western blots prepared. Gels were probed with a rabbit-derived polyclonal antibody directed against K Ca channel protein. The experimental procedure was repeated on separate sets of VSM cells to ensure reproducibility. Expression of K Ca channel protein was diminished in VSM cells incubated with corticosterone 1 carbenoxolone and with RU 28362 after 24 h and remained low at 72 h. Expression of K Ca protein in cells exposed to 11-dehydrocorticosterone 1 carbenoxolone, carbenoxolone alone, and aldosterone was either similar to controls or mildly increased over the 72 h. These data are consistent with the hypothesis that GCs diminish the expression of K Ca protein. Diminished K Ca expression could contribute to the observed increase in vascular tone following chronic GC exposure. © 1999 Academic Press
Blood pressure is regulated by a complex series of physical and hormonal processes. Glucocorticoids have been shown to indirectly or directly alter many of these processes including renal sodium balance and vascular tone (2,5,8,13,14,16,18,24). How glucocorticoids affect the vascular tree has been a subject of intense interest to many investigators given the association of glucocorticoids with hypertension and the widespread use of these steroids in clinical medicine. In humans, both cortisol and synthetic glucocorticoids like dexamethasone increase the sensitivity to applied pressors (18,23). Experiments conducted in various animal and cellular models have confirmed the observation that glucocorticoids have the ability to enhance the contractile response to pressors (4,6,11,12). We have observed that the contractile response of rat aortic rings is enhanced if those rings are incubated with physiologic concentrations of corticosterone and an inhibitor of the enzyme 11b-hydroxysteroid dehydrogenase in vascular smooth muscle which is responsible for local glucocorticoid metabolism (4,6). Moreover, glucocorticoids have been linked to increased hepatic generation of angiotensinogen (9), an increase in angiotensin II type I receptors (21), an upregulation of V1a vasopressin and alpha adrenergic receptors in vascular smooth muscle (15,20), and a decrease in basal guanylate cyclase activity in vascular tissue (25). Calcium-dependent potassium channels con-
1
To whom correspondence should be addressed at Division of Pediatric Nephrology, Rhode Island Hospital, APC 942, 593 Eddy Street, Providence, RI 02903. Fax: (401) 444-3944. E-mail: [email protected]. 53
1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
54
BREM ET AL.
tained in the membrane of vascular smooth muscle cells have recently been shown to play a role in attenuating the vasoconstrictor effects seen in various forms of hypertension (19). When activated, these channels allow for the efflux of potassium from the cell resulting in hyperpolarization and relaxation. To date, there is little in the literature on hormonal control over the expression or activity of this channel. Given the net increase in vascular tone with chronic exposure to glucocorticoids, we hypothesized that these steroids may alter the expression of this form of potassium channel in vascular smooth muscle. In the following studies, we demonstrate that glucocorticoids but not mineralocorticoids have the ability to decrease the expression of potassium channels. METHODS Primary Vascular Smooth Muscle (VSM) Cell Cultures The aortas from eight sacrificed adult SpragueDawley rats were dissected under sterile conditions and associated fat and connective tissue removed. The aortas were next incubated at 37°C for 15 min with digestion mixture which consists of collagenase (1 mg/ml), elastase (0.125 mg/ml), DMEM/F12 (Gibco BRL, Grand Isle, NY) with Hepes (pH 7.4), penicillin (100 mg/ml), and streptomycin (100 mg/ ml). At the completion of the incubation, the adventitia was carefully stripped, the vessel longitudinally opened, and the luminal surface gently scraped with forceps to remove endothelial cells. The remaining tissue was then minced into 1-mm pieces and incubated with digestion mixture for an additional 90 min at 37°C using a shaking water bath. The digestion was stopped with 8 ml of DMEM/F12, 10% fetal calf serum, and 25 mM Hepes. Following digestion, the remnant tissue became a suspension of smooth muscle cells. The cells were passed over a sieve and centrifuged at 1000 rpm for 5 min. Supernatant was discarded and the cells resuspended in DMEM/F12, 10% fetal calf serum, and 25 mM Hepes. The cells were then plated at a concentration of 1 3 10 4 viable cells/cm onto T-25 plates and placed in an incubator. The flasks were gassed with 5% CO 2-95% O 2 and were maintained at 37°C. Cells studied up to 40 passages have shown to retain the expression of a-actin (7), a factor unique to vascular smooth muscle cells (27). Cell counts were obtained before each plating and
prior to any experiment. The medium for the cells was changed twice per week. Studies were usually conducted on the cells after 5 to 7 days when the cells have reached confluence and when they are in a quiescent stage. Cells were made quiescent by removing all growth factors including fetal serum from the incubation medium for 24 h prior to exposure to steroids. Western Blots Vascular smooth muscle cells were grown to confluence in 6-well plates (Corning-Costar, Cambridge, MA) with DMEM/F12 (Gibco BRL) and 10% fetal bovine serum (Sigma Chemical). After reaching confluence, the cells were transferred to serum-free DMEM/F12 for 24 h. At 24 h, the various test compounds were added to the flasks and the incubations were continued for 1 to 3 days. The cells were then rinsed with serum-free DMEM/F12 and scraped off with a cell scraper and 100 mL of running buffer for each well. Protein concentrations were determined with the Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Richmond, CA). Eppendorf tubes containing the samples were boiled for approximately 7 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 8% polyacrylamide gels. Equal amounts of protein were loaded at 100 mg per lane and electrophoretically blotted onto Immobilon-P transfer membranes (Millipore, Bedford MA). The Immobilon membranes were then incubated in TBST (TRIS-buffered saline with 0.05% Tween 20) containing 5% nonfat dry milk (wt/vol) for 30 min at room temperature on a rocking platform, followed by overnight incubation at 4°C in the desired antibody (Bslo K 1 channel rabbit polyclonal antibody) at a 1:250 dilution in fresh TBST-5% nonfat milk buffer. This potassium channel antibody was kindly provided by Dr. Edward Moczydlowski, Department of Pharmacology and Cellular and Molecular Physiology, Yale University School of Medicine. After incubation, the membranes were washed three times for 7 min each in 1X TBST and incubated with HRP-conjugated complementary antibody (Anti Rabbit IgG (whole molecule) peroxidase conjugate, Sigma Chemical, Cat No. A-6154; 1:1000 dilution) in blocking buffer (TBST with 5% nonfat dry milk) for 30 min. The membranes were then washed four times with 1X TBST and exposed to ECL reagent (Amersham Life Science, Buckinghamshire, England) for 1–3 min. The membranes were semidried between two sheets
GLUCOCORTICOIDS INHIBIT K 1 CHANNELS
FIG. 1. A representative Western blot showing the expression of the calcium-dependent potassium channel protein at Day 0 (1 h) and Day 1. For purposes of comparison, the expression of the potassium channel protein from control cells, cells incubated with carbenoxolone (CBX), 1 mM, and cells incubated with the glucocorticoid agonist RU 28362 (RU 362), 1 mM, are shown. Expression of the potassium channel protein was decreased in vascular smooth muscle cells incubated for 24 h with the glucocorticoid RU 28362.
of filter paper and quickly exposed to ECL Hyperfilm (Amersham Life Sciences) for 20 min to 1 h before being developed. The results were analyzed by densitometric scanning using a Power Macintosh running NIH Image 1.60 and expressed as relative increase compared to controls. Where appropriate, results are presented as the density of the experimental sample relative to the density of controls run at the same time under identical conditions. Where appropriate, data were analyzed by Student’s t test with P values less than 0.05 considered significant.
55
hydrogenase inhibitor carbenoxolone alone (1 mM), aldosterone (10 nM), the inactive metabolite, 11dehydrocorticosterone (10 nM) plus carbenoxolone, or the mineralocorticoid antagonist RU 318 (1 mM). From 24 to 72 h, cells exposed to the pure glucocorticoid RU 28362 and cells exposed to corticosterone (10 nM) plus the licorice derivative carbenoxolone (1 mM), which prevents the metabolism of the corticosterone, showed a decrease in the expression of this potassium channel (Fig. 2). The 25 to 30% decrease in potassium channel expression observed with RU 28362 and with corticosterone plus carbenoxolone was not significantly different between 24 and 72 h. As mentioned, vascular smooth muscle cells incubated with aldosterone (10 nM) also showed no clear effect on the potassium channel protein but the expression was considerably more varied. DISCUSSION Glucocorticoid receptors are present in aorta, in mesenteric arteries, and in cultured vascular smooth muscle cells (10). These steroids can bind to and activate these receptors resulting in enhanced vasoconstriction following stimulation with cat-
RESULTS Primary cultures of rat vascular smooth muscle cells were incubated with vehicle or vehicle containing the test reagent(s) for 1, 24, and for 72 h. When the blots at each time point were scanned and the relative density of each blot analyzed compared to its own control, no differences in the expression of the calcium-dependent potassium channel protein were observed at 1 h. As can be seen in a sample Western blot at 24 h, cells exposed to RU 28362 (1 mM) demonstrated a decrease in the expression of the calcium-dependent potassium channel protein (Fig. 1). There was no clear change in the total protein expression relative to control observed in cells previously exposed to the 11b-OH steroid de-
FIG. 2. Scanned densities of the potassium channel Western blots relative to controls. Results are expressed as mean experimental/control 6 SE. Values less than 1 indicate a decreased expression of the potassium channel protein. Since there was no additional change in protein expression after 24 h, the results at 24 h were combined with those at 72 h. Vascular smooth muscle cells appeared to consistently express lower amounts of the potassium channel protein after exposure to RU 28362 [RU362](1 mM) or corticosterone [B](10 nM) plus carbenoxolone [CBX](1 mM). Cells incubated with the inactive metabolite of corticosterone, 11-dehydrocorticosterone [A], plus carbenoxolone did not demonstrate a decreased expression of the potassium channel.
56
BREM ET AL.
echolamines and angiotensin II (18,23). The enhanced constrictor response following chronic glucocorticoid exposure is related, in part, to a documented increased influx of sodium and calcium ions into the vascular smooth muscle cell (12). These changes in intracellular ion concentration may be explained, in part, by a glucocorticoid-induced decrease in the sodium-calcium ion exchanger (22). All these glucocorticoid-induced alterations in membrane permeability fit nicely with the rapidly evolving hypotheses linking changes in intracellular ion concentrations and transport pathways to various forms of hypertension (1,3). Vascular smooth muscle cells also contain several types of potassium channels including the calcium-activated or -dependent potassium channel (17). Activation of this channel leads to an efflux of potassium from the cell, a hyperpolarized transmembrane potential, and vasodilator response. The calcium-dependent potassium channel is activated with various forms of hypertension in an effort to blunt the rise in blood pressure. Thus far, the arachidonic acid derivative 11,12 EET (26) and physical forces like stretch have been shown to directly activate this channel in vascular smooth muscle (17). The current preliminary observations provide evidence that glucocorticoids directly affect the expression of the calcium activated potassium ion channel in vascular smooth muscle. If the downregulated expression of this potassium channel by glucocorticoids translates into a parallel decrease in channel activity, one may expect an attenuated relaxation response and a net rise in vascular tone. The pure glucocorticoid receptor agonist RU 28362 and corticosterone plus carbenoxolone appear to selectively decrease the expression of this potassium channel protein. In contrast, carbenoxolone, an inhibitor of the bidirectional isoform of 11b-OH steroid dehydrogenase found in vascular smooth muscle (7), exerts either no effect or a mildly positive effect on the expression of the channel protein. In addition, vascular smooth muscle cells incubated with 11-dehydrocorticosterone, the inactive product of 11b-OH steroid dehydrogenase, plus carbenoxolone do not show a difference in the expression of this protein relative to controls. The mineralocorticoid aldosterone exerts no clear effect on the expression of the potassium channel protein relative to controls. However, there is a considerable scatter in the data. The steroid-induced downregulation of the potassium channel protein is not immediate; it occurs within 24 h and is maintained to at least 72 h. The
observed time line for the altered channel protein expression is consistent with glucocorticoids directly affecting a process at the nuclear level. The precise process or processes involved with this specific altered protein expression will undoubtedly require further investigation. REFERENCES 1.
Aviv A. The link between cytosolic Ca 12 and the Na 1-H 1 antiport: A unifying factor for essential hypertension. J Hypertension 6:685– 691, 1988.
2.
Baum M, Moe OW, Gentry DL, Alpern RJ. Effect of glucocorticoids on renal cortical NHE-3 and NHE-1 mRNA. Am J Physiol 267:F437–F442, 1994.
3.
Blaustein MP. Sodium ions, calcium ions, blood pressure regulation and hypertension: A reassessment and hypothesis. Am J Physiol 232:C165–C173, 1977.
4.
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