effects of beta-blockers on hmg coa reductase and ldl receptor activity in cultured human skin...

Cardiovascular Drugs and Therapy 1996;10:67-74 © Kluwer Academic Publishers, Boston. Printed in U.S.A.

Effects of Beta-Blockers on and LDL Receptor Activity Skin Fibroblasts

HMG CoA Reductase in Cultured Human

H i r o s h i Yosh ida , M i c h i o S u z u k a w a , T o s h i t s u g u I s h i k a w a , H i d e k i Sh ige , E i s u k e N i sh io , H i r o s h i H o s o a L M a k o t o A y a o r i , a n d H a r u o N a k a m u r a 1st Department of Internal Medicine, National Defense Medical College, Saitma, Japan

Summary. Previous reports, based on clinical trials and animal experiments, suggest that beta-blockers may be useful in the prevention of atherosclerosis. Betaxolol, a new betal- selective blocker, was shown to decrease plasma total and LDL cholesterol levels or to have no adverse effect on those [1-4]. While many reports deal with the metabolism of tri- glyceride and high density lipoprotein, fewer publications about cholesterol metabolism are currently available. To clarify the mechanism by which beta-blockers affect lipid metabolism, we examined the effects of beta-blockers on HMG CoA reductase and LDL receptor activity in cultured human skin fibroblasts. L-propranolol, a nonselective beta- blocker, increased HMG CoA reductase activity and decreased LDL receptor activity. However, d-propranolol had no major effects on HMG CoA reductase activity. These re- suits suggest that beta-blockers act on HMG CoA reductase through the beta receptors. Betal-blocking action should decrease HMG CoA reductase activity and increase LDL receptor activity. In fact, betaxolol, a betal-selective blocker, decreased HMG CoA reductase activity and increased LDL receptor activity, but metoprolol had no major effect. We speculate that the discrepancy between betaxolol and metoprolol in the effect on HMG CoA reductase and the LDL receptor might be due to the difference of the extent of betal- selectivity. We conclude that betal-selective blockers are antihypertensive agents potentially valuable in the prevention of atherosclerosis.

Cardiovasc Drugs Ther 1995;10:67-74

Key Words. HMG CoA reductase, LDL receptor, beta- blocker, betal-selectivity, human skin fibroblasts

cemia [7]. In fact, treatments with diuretics and some beta-blockers have been reported to increase plasma triglycerides (TG) and to decrease high density lipoprotein (HDL) cholesterol [7,8]. However, beta 1- selective blockers or partial agonistic beta-blockers have been reported to have no significant adverse effect on plasma lipids [9,10].

Several large studies (e.g., Lipid Research Clinics Intervention Trial [11], Helsinki Heart Study [12]) point out the beneficial effects of treating dyslipidemia in preventing coronary heart disease. It is possible that slight effects of antihypertensive drugs on lipid metabolism influence the risk of coronary heart disease.

Among the usual antihypertensive drugs, nonselective beta-blockers without partial agonistic activity (PAA), such as propranolol, tend to cause the most pronounced changes, increasing TG and decreasing HDL cholesterol. These alterations are slightly less pronounced with selective beta-blockers without PAA and with partial agonistic beta-blockers. The mecha- nisms by which beta-blockers exert their effects on lipoprotein metabolism are only partially understood. Beta blockage is accompanied by increased alpha- adrenergic tone, which is known to lower lipoprotein lipase activity and then results to increased TG levels due to an impaired catabolism of TG-rich lipoprotein [13]. Thus, HDL, which depends on very low density lipoprotein (VLDL) catabolism, decreases, as indi-

Hypertenion is a major risk factor in cardiovascular disease. Several studies [5,6] reported that a reduction of blood pressure in hypertensive patients leads to a decrease in morbidity and mortality from stroke and other hypertensive sequellae, but failed to indi- cate a significant decrease in the incidence of coronary heart disease. It is now considered very important for antihypertensive drugs to not only control blood pressure but also to prevent metabolic disorders, such as dyslipidemia, glucose intolerance, and hyperuri-

This paper was partially presented at the llth Drug Affecting Lipid Metabolism Symposium in Italy in 1992 and at the 6th American Hypertension Symposium in New York in 1991. Address for correspondence: Hiroshi Yoshida, MD, 1st Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359 Japan.

Received 25 June 1995; receipt~review time 2 months, 17 days; accepted in revised form 13 September 1995.

67

68 Yoshida et al.

cated by a close inverse relationship between beta- blocker induced changes in TG and HDL cholesterol.

On the other hand, the effects of beta-blockers on cholesterol metabolism are not entirely clear. In our clinical trial, betaxolol, a betal-selective blocker, decreased total and LDL cholesterol levels in a group that initially had higher plasma levels of total and LDL cholesterol [4]. Brown et al. [14] demonstrated an inverse relationship between total and LDL cholesterol levels and LDL receptor activity in liver. Subse- quently, Brown and Goldstein [15] described a LDL receptor pathway that regulates cholesterol homeostasis in cells by operating through an LDL receptor, 3-hydroxy-e-methylglutaryl coenzyme A (HMG CoA) reductase, a rate-limiting enzyme of cholesterol bio- synthesis, and acyl coenzyme A cholesterol acyltransferase (ACAT) activity. These activities are affected by genetic, nutritional, hormonal, and pharmacologi- cal factors. Goldstein et al. [16] and Suzukawa and Nakamura [17] suggested that the suppression of HMG CoA reductase activity could upregulate LDL- receptor activity.

The mechanism by which beta-blockers affect cholesterol metabolism is unknown. To clarify the effects of beta-blockers on plasma lipids, and especially cholesterol, we examined the effects of beta-blockers on HMG CoA reductase and LDL receptor activity in cultured human skin fibroblasts.

M a t e r i a l s a n d M e t h o d s

DL-3-(glutaryl-3-14C)-hydroxy-3-methylglutaryl CoA (51.6 mCi/mmol), RS-[5-3H(N)] mevalonolacton (28.4 Ci/mmol), and Omnifluor were obtained from New England Nuclear (Boston, MA). Brij 96, potassium phosphate, ghcose-6-phosphate, dithiothreitol, beta- nicotinamide adenosine dinucleotide phosphate, and glucose-6-phosphate dehydrogenase were purchased from Sigma Chemical (St. Louis, MO). Silica gel thin layer chromatography plates were obtained from E. MERCK (Strasse Frankfurt). Tissue culture flasks and dishes were supplied by Miles Laboratories (Na- perville, IL). Dulbecco's modified Eagle's medium (DMEM) and Dulbecco's phosphate buffered saline were purchased from Flow Laboratories (Irvine, Scotland). Penicillin (10,000 units/ml)-streptomycin (10,000 ~g/ml), and fetal calf serum were supplied by Gibco Laboratories (Grand Island, NY). 125I-iodine was obtained from Amersham (Arlington Heights, IL). Iodo-gen (1,3,4,6-tetrachloro-3a-5a-diphenyl- glycuril) was purchased from Sigma Chemical. Biogel P-6 was obtained from Bio-rad Laboratories (Rich- mond, CA). HEPES [N-(2-hydroxy-ethyl)-piperazine- n-2-ethane-sulfonic acid] was supplied by Nakarai Tesque (Kyoto). Betaxolol was a gift from Mitsubishi Kasei (Tokyo). L-propranolol, d-propranolol, metoprolol, and salbutamol were obtained from Sigma Chemical (St. Louis, MO).

Cell cultures The normal human skin fibroblasts used came from a skin biopsy of a normolipidemic adult man. Cells were grown in a monolayer for 7-15 generations prior to use. Cell lines were maintained in a humidified C02 incubator at 37°C in 75 cm ~ flasks containing 10 ml DMEM supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% (V/V) fetal calf serum. Cells from the stock flasks were dissociated with Dulbecco's phosphate buffered saline (PBS) containing 0.25% trypsin and 0.05% EDTA, and were seeded at approximately 2 × 104 cells per dish (60 mm) containing 3 ml DMEM with 10% fetal calf serum. On day 3, the medium was replaced with fresh medium and on day 6 (before the cells reached conflu- ence) the medium was removed.

The monolayer of cells was washed twice with 2 ml PBS, and was changed to DMEM with 10% lipoprotein deficient serum (LPDS). LPDS (d > 1.215 g/l) was prepared by ultracentrifugation of normal human serum followed by 48 hours of dialysis against 150 mM sodium chloride [16,17]. After incubation for 48 hours, beta-blockers dissolved in distilled water were added to the medium to reach the predetermined concentrations, and the cells were incubated for 14 hours. The beta-blockers used and the predetermined concentrations were as follows: the nonselective beta-blockers, 1-propranolol and d-propranolol, at concentrations of 25, 50, 100, and 200 ng/ml and at 25, 50, and 100 ng/ ml, respectively; the betal-selective beta-blockers, metoprolol and betaxolol, at concentrations of 50 and 100 ng/ml, and at 20, 40, and 60 ng/ml, respectively. Salbutamol was used at the concentrations of 5-20 p.M.

Measurement of HMG CoA reductase activity HMG CoA reductase activity was measured using the technique of Goldstein et al, [16]. After discarding the medium from each dish, the cells were washed twice with PBS and harvested with a rubber policeman, and the cell pellets were then stored at -80°C until use. Cell-free extracts were prepared by suspending the pellets in 80 ~l of 50 mM potassium phosphate buffer (pH 7.4) containing 5 mM EDTA, 200 mM potassium chloride, and 0.25% brij 96. After incubation for 30 minutes at 37°C, the suspension was centrifuged for 15 minutes at 12,000 × g and the supernatant was incubated at 37°C for 60 minutes in a final volume of 50 ~1 containing 100 mM potassium phosphate (pH 7.5), 20 mM giucose-6-phosphate, 2.5 mM NADP, 4 mM dithiothreitol, 0.5 units glucose-6-phosphate dehydrogenase, and 30 ~M DL-3-[3-14C]-HMG CoA (51.6 mCi/mmol). The reaction was terminated by adding 10 ~1 of 2 M HCI.

The mixture was further incubated at 37°C for 10 minutes in order to convert mevalonic acid to mevaionolacton. After adding [3H]-mevaionolacton (100,000 cpm) as an internal standard to correct for incomplete recovery of mevalonolacton, the mixture was chro-

Effects of Beta-Blockers on HMGCoA Reductase 69

matographed on a thin layer plate of silica gel G using a solvent mixture of acetone/benzene (1/1, v/v). The area containing [14C] mevalonolacton was scraped off and placed in a vial containing 5 ml scintillation fluid (Omnifluor 8 g/l, dioxane: toluene = 4:1). Radioactiv- ity was measured by a liquid scintillation counter, and cell proteins were measured by the method of Lowry et al. [18] using bovine serum albumin as the standard.

Measurement of lipids content in cells Total cell lipids were extracted by the method of Bligh and Dyer [19]. Cellular lipids were assayed enzymati- cally by the method of Heider and Boyett [20] and Suzukawa et al. [21].

Lipoprotein preparation and radioiodination Low density lipoprotein in the density range 1.019-1.063 was isolated by ultracentrifugation from normolipidemic human plasma [22] and was dialyzed against a buffer containing 150 mM sodium chloride and 0.24 mM EDTA, pH 7.4, for 24 hours at 4°C. After dialysis, the LDL solution was sterilized by pas- sage through a Millipore filter (Millex-HA, 0.45 ~m). The LDL was radioiodinated using carrier-free Na 125I and Iodo-gen [17,22,23]. Iodo-gen (1 mg) was dissolved in 1 ml of diethyl ether, and 100 ~l of the solution was dispersed in the bottom of a glass tube and evaporated to dryness at room temperature under ni- trogen. This produced a film of Iodo-gen that was not mixed with buffer. The LDL solution (500 ~g protein in 100 ~l) was added to the iodination tube, followed by Na 125I (1 mCi, 10 }xl). The iodination was allowed to proceed for 15 minutes at 4°C, ensuring that the reactants were in contact with the Iodo-gen film at the bottom of tube.

The whole mixture was applied to a Bio-gel P-6 column (1-25 cm). After eluting the void volume with PBS, the initial 2 ml eluate was collected and dialyzed against 6 1 of buffer (150 mM sodium chloride and 0.24 mM EDTA, pH 7.4) for 48 hours. The specific activity of 125I-LDL was 150 cpm/ng protein and was not changed after delipidating this LDL. Iodo-gen treated LDL did not show any change in electrophoretic mo- bility on agarose gel electrophoresis. Therefore, this Iodo-gen treated LDL was not modified oxidatively.

Measurement of binding and internalization of 125I Binding and internalization of 125I-LDL was measured according to the techniques of Goldstein et al. [16]. After incubation for 14 hours in the medium containing the indicated drugs, 10 ~Lg protein of 125-I- LDL, in the presence or absence of 500 ~Lg of unlabeled LDL, was added to the medium, and the cells were incubated for 4 hours at 4°C (for binding assay) or 37°C (for internalization assay). After the medium had been removed, the cells were rapidly washed three times with ice-cold buffer A (150 mM NaC1, 50

mM Tris-HC1, 2 mg/ml bovine serum albumin, pH 7.4). After washing twice more for 10 minutes with buffer A at room temperature, the cells were im- mersed in buffer B (50 mM NaC1, 10 mM HEPES, 5 mg/ml heparin, pH 7.4) and placed on a shaker for 60 minutes at 4°C. The buffer B was collected and an aliquot was radiocounted for I~SI-LDL released from the cell surface.

The cells were dissolved by standing for 1 hour at room temperature in 0.1 M NaOH. One aliquot was radiocounted to determine the amount of 125I-LDL that had been internalized by the cells, and another aliquot was used to determine the cellular protein content. Data are expressed as nanograms of LDL bound or internalized per milligram of cell protein during 4 hours of incubation. Scatchard analysis of I~I-LDL bound to the cells was performed under incubation for 4 hours at 4°C at concentrations of 2-100 ~Lg/ml of 12~I-LDL in the medium, respectively, in the presence or absence of a 50-fold excess of unlabeled LDL.

Statistical analysis Values are expressed as the mean -+ SD. The signifi- cance of differences between mean values was deter- mined by ANOVA followed by two-tailed Duncan's test analysis for multiple comparisons. A value of p < 0.05 was accepted as significant [24].

Resu l t s

Effects of beta-blockers on HMG CoA reductase activity in cells HMG CoA reductase activity in human fibroblast cells was significantly increased by the addition of 1- propranolol to the culture medium at concentrations greater than 50 ng/ml (Figure 1A). On the other hand, d-propranolol at concentrations up to 100 ng/ml had no major effect on HMG CoA reductase activity (Fig- ure 1B). Addition of metoprolol, a betal-selective blocker, to the culture medium had no significant effect on HMG CoA reductase activity (Figure 2A). However, betaxolol, another betal-selective blocker, significantly decreased HMG CoA reductase activity at concentrations greater than 20 ng/ml (Figure 2B).

Effects of beta-blockers on cellular l o id s At higher concentrations, two beta-blockers (1-propranolol and betaxolol) significantly affected cellular cholesterol levels in cultured fibroblasts, while the other two (d-propranolol and metoprolol) had no dis- cernible effect (Table 1). 1-propranolol at a concentration of 100 ng/m| increased total (TC) and free (FC) cholesterol levels in cells, while betaxolol at 60 ng/ml had the opposite effect (Table 1).

Effects of beta-blockers on LDL receptor activity At concentrations of 20-60 ng/ml, betaxolol increased LDL binding and internalization in a dose-dependent

70 Yoshida et al.

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Fig. 1. Effects of nonselective (l-propranolo1, d-propranolol) beta-blockers on HMG CoA reductase activity in cultured human skin fibroblasts. After incubation for 14 hours with the indicated concentrations of 1-propranolol (A) or d-propranolol (B), HMG CoA reductase activity was measured as described under Materials and Methods. Data are expressed as pmol/min/mg cell protein. Values represent the mean +- SD of (A) or (B) dishes in each group.

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Fig. 2. Effects of betal-selective (metoprolol, betaxolol) beta-blockers on HMG CoA reductase activity in cultured human skin fibroblasts. After incubation for 14 hours with the indicated concentrations of metoprolol (A) or betaxolol (B), HMG CoA reductase activity was measured as described under Materials and Methods. Data are expressed as pmol/min/mg cell protein. Values represent the mean +- SD of five dishes in each group.

manner (Table 2). Scatchard analysis of LDL binding data indicated that the values of Bmax (ng of LDL protein bound/mg cell protein) were 39.5 and 52.1, and for Kd (l~g of LDL protein/ml) were 10.94 and 11.08 in cells treated with nothing (control) and betaxolol (60 ng/ml), respectively. The upregulation of LDL receptor activity is due to an increase in the number of LDL receptors with no change in binding affinity (Figure 3). In contrast, 1-propranolol had a negative effect on LDL receptor activity (Table 3). The decrease is presumed to be associated with the increased HMG CoA reductase activity induced by 1-propranolol. On the other hand, d-propranolol had just the opposite effect; it tended to increase LDL receptor activity but not significantly (Table 3).

E f f e c t o f b e t a 2 - s t i m u l a t i n g on H M G C o A reductase activity Figure 4 shows that salbutamol, a beta2-stimulant, tends to decrease HMG CoA reductase activity in cultured fibroblasts in a dose-dependent fashion and significantly decreases that activity at the concentration of 20 I~M. Moreover, it is interesting that salbutamol reduces the increase of HMG CoA reductase activity in cultured fibroblasts co-incubated with 1-propranolol (Figure 5).

Discussion Cultured human skin fibroblasts have been shown to have on their surface specific high affinity receptors that bind LDL [25]. These receptors appear to be reg-


Table 1. Effects of beta-blockers on cellular lipids

TC FC TG

Control 43.19 ± 2.99 32.13 ± 2.28 33.31 ± 3.74 L-propranolol

50 ng/ml 46.61 ± 4.01 34.23 ± 4.55 35.51 ± 3.09 100 ng/ml 48.88 ± 2.18 a 37.85 ± 2.27 b 35.22 _+ 3.05

D-propranolol 50 ng/ml 43.56 -+ 3.91 31.93 +- 2.04 33.51 _+ 2.91

100 ng/ml 42.82 ± 2.95 31.29 ±- 2.02 34.58 ± 3.66 Betaxolol

20 ng/ml 42.84 _ 2.39 31.04 +- 2.19 35.78 ± 2.73 40 ng/ml 40.04 ± 4.85 30.41 ± 2.92 35.69 ± 2.84 60 ng/ml 37.74 ± 1.615 27.03 ± 1.925 35.22 ± 2.56

Metoprolol 50 ng/ml 41.76 +- 3.89 33.66 ± 5.31 36.34 +- 3.61

100 ng/ml 44.67 ± 4.31 32.68 ± 3.17 36.64 ± 3.12

Units: I~g/mg cell protein (mean -+ SD). a0.05 < p value < 0.1 (compared with control). b0.02 < p value < 0.05 (compared with control). After incubation for 14 hours with the indicated concentrations of beta-blockers, cellular lipids were measured as described under Ma- terials and Methods. Data are expressed as ~g/mg cell protein. Val- ues represent the mean ~ Sd of quadruplicate dishes in each group.

Table 2. Effects of increasing concentrations of betaxolol on binding and internalization of I~SI-LDL in cultured human skin fibroblasts

Betaxolol (ng/ml) 0 20 40 60

Binding Specific 539.2 720.6 ~ 870.2 a 987.95

Nonspecific 217.4 256.8 206.8 198.8 Internalization

Specific 2240.4 2692.6 a 3002.4 a 3386.15 Nonspecific 341.7 388.1 433.9 396.6

ap < 0.05; bp < 0.01 as compared with betaxolol 0 ng/ml. Values for specific binding and internalization are equal to values for total minus nonspecific binding and internalization. Total binding and internalization were assayed in quadruplicate determinations, and nonspecific binding and internalization were assayed in duplicate determinations. Values represent the mean of these determinations. Data points are expressed as ng/mg cell protein.

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Fig. 3. Kinetics of LDL binding to surface receptors of fibroblasts. The figure represents a recalculated Scatchard plot of the actual binding data obtained from triplicate determinations. Recalculated data were f t ted by least squares regres- sion analysis.

Table 3. Effects of propranolol on binding and internalization of I~SI-LDL in cultured human skin fibroblasts

Binding Internalization

Propranolol Specific Specific (ng/ml) Nonspecific Nonspecific

1-propranolol(100) 486.2 140.1 a 2086.8 390.4 a d-Propranolol (100) 631.1 132.6 a 2418.9 355.5 a Control 556.2 (170.3) 2244.9 352.7

#0.05 < p < 0.1, as compared with control. Values for specific binding and internalization are equal to values for total minus nonspecific binding and internalization. Total binding and internalization were assayed in quadruplicate determinations, and nonspecific binding and internalization were assayed in duplicate determinations. Values represent the mean of these determinations. Data points are expressed as ng/mg cell protein.

ulated by a feedback mechanism responsive to the availability of LDL in the surrounding medium. The agents compactin and mevinolin, isolated from various molds, are competitive inhibitors of HMG CoA reductase [26,27]. Inhibition of cholesterol synthesis leads to a reduction of free cholesterol content in cells and thereby to the responses of an increased amount of HMG CoA reductase mRNA and LDL receptor mRNA [28, 29]. By this mechanism, the increased LDL receptor activity lowers plasma LDL cholesterol levels.

In a clinical trial, betaxolol, a new betal-selective

blocker, decreased plasma total and LDL cholesterol levels when the initial values of plasma total and LDL cholesterol were relatively higher [4]. Previous reports [1,2] showed that betaxolol, in contrast with other beta-blockers, has no adverse effect on lipid metabolism and carbohydrate metabolism. These effects of betaxolol are considered to be more highly betal- selective than are other beta-blockers. McAreavy et al. [3] reviewed the new beta-blockers and the treatment of hypertension, and they reported that betaxolol decreases LDL cholesterol levels. We thought that betaxolol might lower plasma LDL cholesterol levels

72 Yoshida et al.

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Fig. 4. Effects of salbutamol on HMG CoA reductase activity in cultured human skin fibroblasts. After incubation for 14 hours with the indicated concentrations of salbutamol, HMG CoA reductase activity was measured as described under Ma- terial and Methods. Data are expressed as pmol/min/mg cell protein. Values represent the mean +_ SD of 5 dishes in each group. *p < 0.05.

by the inhibition of HMG CoA reductase. However, betaxolol did not directly inhibit HMG CoA reductase activity in the cell-free extract (data not shown) and suppressed HMG CoA reductase activity in cultured fibrobasts (Figure 2B). Thus, betaxolol is not a competitive inhibitor of HMG CoA reductase.

We considered that beta-blockers might act on HMG CoA reductase activity through beta-receptors. 1-Propranolol increased HMG CoA reductase activity (Figure 1A) but d-propranolol had no major effect on HMG CoA reductase activity (Figure 1B). These results suggest that beta-blockers act on HMG CoA reductase activity through the beta-receptor, since d- propranolol has lesser beta-blocking effects than 1-propranolol. However, this paper has the limitations of these observations since we have not measured the amount of beta-receptors in cultured human skin fibroblasts. It is interesting that 1-propranolol increased HMG CoA reductase activity (Figure 1A), contrary to the effect of betaxolol (Figure 2B). Be- cause propranolol is a nonselective beta-blocker, we thought that betae-blocking action could increase HMG CoA reductase activity.

Figure 4 shows that salbutamol, a beta2-stimulant , decreased HMG CoA reductase activity at 20 ~M in cultured human skin fibroblasts and reduced the upregulation of HMG CoA reductase activity with l- propranolol (Figure 5). Metoprolol, a betal-selective blocker, had no major effect on HMG CoA reductase activity (Figure 2A), contrary to the effect of betaxolol (Figure 2B). It is very likely that the difference between metoprolol and betaxolol effects on HMG CoA reductase activity can be attributed to a difference in the level of betal-selectivity; betaxoM is more strongly betal-selective than metoprolol [30]. There-

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Fig. 5. Effect of coincubation of salbutamol and l- propranolol on HMG CoA reductase activity in cultured human skin fibroblasts. After incubation for 14 hours with the indicated concentration of salbutamol and l-propranolol, HMG CoA reductase activity was measured as described under Materials and Methods. Data are expressed as pmol/min/ mg cell protein. Values represent the mean +_ SD of five dishes in each group. *p < 0.05 A; control, B; l-propranolol 100 ng/ml, C; 1-propranolol 100 ng/ml and salbutamol 20 ~ , D; l-propranolol 200 ng/ml, E; l-propranolol 200 ng/ml and salbutamol 20 tdi/I.

fore, it may be that betal-receptor pathway could act by increasing HMG CoA reductase activity and the beta2-receptor pathway could act by decreasing its activity.

By this mechanism, betal-blocking action could increase LDL receptor activity and tend to reduce plasma cholesterol levels; on the contrary, beta e- blocking action could decrease LDL receptor activity and tend to raise plasma cholesterol levels. However, this research study is limited by these observations since we have not specified the amount of the different kinds of beta-receptors in human skin fibroblasts. The effects and differences between betaxolol and meto- prom might be due to differences in the membrane- stabilizing activity or lipid solubility.

Table 1 shows that 1-propranolol increased, but betaxolol decreased, cellular cholesterol levels. Since the cells were incubated in lipoprotein-deficient medium, we suggest that cellular cholesterol levels change secondarily parallel to the change of HMG CoA reductase activity. The inhibition of HMG CoA reductase leads to decreased cellular cholesterol levels in the lipoprotein-deficient medium and results in the upregulation of LDL receptor activity. On the other hand, stimulation of HMG CoA reductase activity leads to increased cellular cholesterol levels and results in the downregualtion of LDL receptor activity. Cells are preincubated for 48 hours in lipoprotein- deficient medium. This is a maximum stimulus to LDL receptor expression.

In this condition, betaxolol and 1-propranolol show


a measurable effect. This should imply the ability of the drugs to modulate their targets independent of the effects mediated by cholesterol. In fact, the data on cellular cholesterol are not always consistent with the data on LDL receptor activity and HMG CoA reductase activity (Tables 1 and 2; Figure 2). Betaxolol modulates HMG CoA reductase activity and LDL receptor activity at 20 ng/ml, but has no major effect on cellular cholesterol level. This inconsistency might be due to the time lag between the decrease of cellular cholesterol level and the decrease of HMG CoA reductase activity. 1-Propranolol increased HMG CoA reductase activity (Figure 1A) and reciprocally decreased LDL receptor activity (Table 3). In contrast, betaxolol decreased HMG CoA reductase activity (Figure 2B) and reciprocally increased LDL receptor activity (Table 2). The upregulation of LDL receptor activity with betaxolol is due to the increased LDL receptor number, with no major change of binding affinity (Figure 3).

Interestingly, d-propranolol tended to increase LDL receptor activity (Table 3), even though d-propranolol had no major effect on HMG CoA reductase activity (Figure 1B). Although this effect of d-propranolol was insignificant, the mechanism by which d-propranolol tended to increase LDL receptor activity remains a mystery. To clarify the mechanism of upregulation of LDL receptor activity with d-propranolol, we investigated Scatchard analysis of LDL binding in fibroblasts incubated with d-propranolol. d-Propranolol did not change LDL receptor number, and tended to increase the binding affinity, but not significantly (data not shown). This problem requires further investigations (e.g., LDL receptor protein levels, LDL receptor mRNA levels).

These data regarding the effect of propranolol on HMG CoA reductase and LDL receptor activity agree with the general concept that in clinical administration, propranolol has no major effect on plasma LDL cholesterol levels because 1,d-propranolol is utilized to treat hypertension, angina, and so on. Mazi~re et al. [31] and Bernini et al. [32] reported that propranolol increased HMG CoA reductase activity and LDL receptor activity. However, the concentrations of drugs used in their studies were higher than plasma levels of drugs measured in clinical doses. Thus, the difference in conclusions probably stems from differences in the concentrations of drugs used in the studies. We suggest that the amount of drug in the medium is an important factor in studying the effects of drugs on lipid mediators in vitro. In fact, our preliminary data showed that 1-propranolol increased both HMG CoA reductase activity and LDL receptor expression at 1-1000 ~g/ml, and these data agree with previous reports.

Supporting evidence for the antiatherosclerotic effects of betal-selective blockers is provided by a number of published animal experiments. Metoprolol treatment significantly reduced atherosclerotic lesions

of the aorta in animals fed an atherogenic diet [33,34]. In the MAPHY study, metoprolol treatment lowered the risk for coronary events [35,36]. Based on the evidence in these reports and in our study, we suggest that betal-highly selective blockers, such as betaxolol, may reduce the risk of atherosclerosis more effec- tively than do other beta-blockers, aside from the condition of partial agonistic activity.

In conclusion, beta-blockers act on HMG CoA reductase and LDL receptor activity through beta- receptors in cultured fibroblasts. It is possible that betal-highly selective blockers decrease HMG CoA reductase activity and reciprocally increase LDL receptor activity. Therefore, betal-selective blockers should be valuable agents in the prevention of atherosclerosis.

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obese hypertensive patients. Long-term effects on blood pressure and serum lipids. Neth J Med 1992;40:227-231.

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effects of beta-blockers on hmg coa reductase and ldl receptor activity in cultured human skin...

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