Reduction of protein degradation and atrophy cultured fetal mouse hearts by leupeptin PETER
LIBBY,
JOANNE
S. INGWALL,
AND
ALFRED
in
L. GOLDBERG of Medicine,
Department of Physiology, Harvard Medical School, and Department Peter Bent Brigham Hospital, Boston, Massachusetts 02115 LIBBY, PETER, JOANNE S. INGWALL, AND ALFRED L. GOLDBERG. Reduction of protein degradation and atrophy in cultured feta2 mouse hearts by Zeupeptin. Am. J. Physiol. 237(l): E35-E39,1979 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 6(l): E35-E39, 1979.-This study examined the effects of certain protease inhibitors on protein turnover, atrophy, and viability of cultured fetal mouse hearts. Leupeptin (30 PM) diminished net proteolysis by about 50% (P < 0.001) and as a consequence retarded cardiac atrophy. Hearts cultured with leupeptin for 2 days contained 19% more protein than control hearts (P < 0.02). Leupeptin did not alter microscopic appearance, pattern of contraction, rates of protein synthesis and protein leakage, or levels of adenosine triphosphate, lactate dehydrogenase, and creatine kinase. Other protease inhibitors (antipain, pepstatin, chymostatin, and those from soybean and bovine lung) had little or no effect on proteolysis and did not decrease atrophy. Leupeptin inhibits intracellular cathepsin B activity. However, in hearts exposed to leupeptin for 48 h and then washed to remove the inhibitor, cathepsin B level was twice control, whereas three other lysosomal hydrolase activities changed little or not at all. Thus, prolonged exposure to its inhibitor selectively increased tissue content of this lysosomal protease. These findings illustrate the importance of protein breakdown in determining tissue mass. Leupeptin appears useful in studies of protein turnover, in maintaining tissues or organs in culture, and possibly in the therapy of certain diseases. protein breakdown; cardiac muscle
cathepsin
B; protease
inhibitors;
lysosomes;
RATE of protein breakdown in a tissue is an important determinant of its protein content (6, 7,22, 31). Agents that specifically reduce this process could prove useful in investigations of the mechanism, control, and physiological role of intracellular protein breakdown. Because such agents should theoretically alter tissue growth or atrophy, they could also be useful in treatment of pathological states in which proteolysis is accelerated and in the maintenance of tissues and organs in culture. The protease inhibitor leupeptin decreases proteolysis in skeletal and atrial muscle (12) and isolated liver cells incubated in vitro for several hours (9, 11). Because cathepsin B activity is diminished in homogenates of leupeptin-treated muscles (12) and hepatocytes (unpublished observations), this lysosomal protease may be one site of the action of leupeptin. Previous reports have also suggested that mixtures of protease inhibitors containing leupeptin might be useful in preventing degeneration of muscles in animals with muscular dystrophy (15, 16, 24).
THE OVERALL
0363-6100/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
However the effective agent, its mechanism of action, and potential toxic effects of long-term exposure to protease inhibitors have not been investigated. Therefore this study investigated whether prolonged exposure to leupeptin reduces net proteolysis and protein loss from an atrophying tissue. These experiments also determined whether exposure to this agent for 2 days. produced toxic effects or altered the activities of lysosomal hydrolases. The fetal mouse heart offered several advantages for such a study. The hearts continue to beat for several days when incubated in defined media (10, 30). They atrophy markedly in vitro (10,30), perhaps because they do not contract against a load. Thus, this preparation permitted long-term observations under controlled conditions and allowed us to test the hypothesis that a protease inhibitor might selectively reduce protein loss from a differentiated tissue in negative nitrogen balance. MATERIALS
AND
METHODS
Chemicals and supplies. The protease inhibitors leupeptin, antipain, chymostatin, and pepstatin, peptide derivatives produced by actinomycetes (27), were kindly supplied by Prof. H. Umezawa (Tokyo, Japan). Bovine lung protease inhibitor (aprotonin, or Trasylol) was manufactured by Boehringer Mannheim Biochemicals (Indianapolis, IN). [3H]phenylalanine (7 mCi/mM) was purchased from Schwartz/Mann (Orangeburg, NY). Carbobenzoxy-alanyl-arginyl;arginyl-4-methoxy-2-naphthylamine and 4-methoxy-2-naphthylamine were supplied by Enzymes Systems Products (Livermore, CA). Soluene was purchased from Packard Instrument Co. (Downs Grove, IL). [ “C]methylglobin was prepared as previously described (20). Vitamins and antibiotics were bought from Grand Island Biochemical Co. (Grand Island, NY), and Falcon organ culture plates were bought from Becton-Dickenson & Co. (Oxnard, CA). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Organ Cultures. Hearts from fetuses of CD-l mice 1617 days postconception (Charles River Breeding Laboratories, Wilmington, MA) were incubated on a stainless steel screen at the medium/gas interface of organ culture plates, as previously described (29). Generally, two hearts were incubated per plate. The medium was Krebs-Ringer bicarbonate buffer, supplemented with glucose (10 mM) and insulin (0.1 U/ml) except as noted. All media were equilibrated with 95% 02: 5% CO2 and contained penicillin (100 U/ml), streptomycin 10 (pg/ml), and amphoterecin B 0.25 @g/ml). Society
Downloaded from www.physiology.org/journal/ajpendo at Macquarie Univ (137.111.162.020) on February 13, 2019.
E35
LIBBY,
E36
Net protein
degradation.
Net proteolysis, i.e., the balance between protein synthesis and degradation, was determined by measuring the release of tyrosine into the incubation medium (4, 5). Because this amino acid is neither synthesized nor degraded by cardiac muscle, its release into the medium reflects the net degradation of tissue proteins to amino acids (4, 5, 18)‘ The release of tyrosine underestimates the actual rate of protein breakdown because some of the tyrosine released by protein hydrolysis is reincorporated during protein synthesis. In these experiments we have ignored possible changes in the intracellular pools of tyrosine because these pools in cardiac muscle are much smaller than the amounts appearing in the medium in these long-term incubations (18)
Piotein synthesis and adenosine surements. After 48 h of incubation
triphosphate
mea-
in medium with or without leupeptin, hearts were incubated for 3 h with or without the inhibitor in Krebs-Ringer bicarbonate buffer supplemented with glucose (10 mM), insulin (0.1 U/ml), 5 times rat plasma concentrations of amino acids (15), except for phenylalanine (which was included at 0.5 mM final concentration), vitamins for minimal essential medium, and [3H]phenylalanine (15 &i/plate). Each heart was then immediately placed in cold perchloric acid (0.2 M), and homogenized, and 2 mg of bovine serum albumin (10% wt/vol in water) were added. After centrifugation, the pellet was washed twice in perchloric acid, once in ethanol-ethyl ether (1: 1 vol/vol), dissolved in Soluene (0.4 ml), and counted as described (6). Samples of the media were also counted. Efficiency of counting was determined by use of an automatic external standard. The rate of phenylalanine incorporation was calculated assuming that the specific activity of phenylalanine in the medium approximated that of the precursor pools for protein synthesis. This assumption is probably valid for hearts incubated with 0.5 mM unlabeled phenylalanine (17). The perchloric acid supernatant of these hearts was neutralized with potassium hydroxide and the volume noted. After centrifugation at 4°C to remove potassium perchlorate, the adenosine triphosphate concentration was determined in the supernatant photometrically using fnefly luciferase (23). Protein and enzyme assays. Protein was determined by the method of Lowry et al. (13) with bovine serum albumin as the standard. To measure protein released into the incubation medium, aliquots of media were pooled and treated with perchloric acid (final concentration 0.2 M) to precipitate proteins prior to assay. Cathepsin B was measured using the fluorogenic substrate carbobenzoxy-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine (12, 25), after washing the hearts to remove the inhibitor (Table 4). N-acetyl-P-glucosaminidase and acid phosphatase were measured calorimetrically by standard techniques (2). The leupeptin-insensitive acidic proteolytic activity was measured by incubating [‘“Clmethylglobin in sodium acetate buffer (0.1 M, pH 4) in the presence of leupeptin (3 PM). The release of radioactivity soluble in perchloric acid (0.2 M) was measured. Lactate dehydrogenase (3) and creatine kinase (21) were measured spectrophotometrically. All statistical calculations used Student’s t test for independent observations.
INGWALL,
AND
GOLDBERG
RESULTS
The fetal mouse hearts were in marked negative nitrogen balance. Net protein degradation occurred at a constant rate during 48 h of incubation, as indicated by the linear release of tyrosine from the tissue (Fig. 1, Table 1). At all concentrations tested, leupeptin reduced the rate of net protein breakdown (Fig. 1, Table 1). This agent decreased net protein breakdown to a similar extent in hearts incubated in medium supplemented with glucose alone, and those incubated in medium further enriched with insulin (0.1 U/ml) and 5 times normal rat plasma levels of amino acids (data not shown). Because mixtures of protease inhibitors have been reported to delay degeneration of dystrophic skeletal muscle (15, 16, 24), we also tested whether a variety of additional protease inhibitors also altered net protein breakdown. The compounds tested were antipain, pepstatin, and chymostatin, which are other peptide aldehydes produced by actinomycetes (27), and the polypeptide soybean trypsin inhibitor and bovine lung protease inhibitor (Trasylol or aprotonin). Of these compounds only antipain decreased net proteolysis in the cultured hearts, but its effect was much smaller than that of leupeptin (Table 2). The fetal mouse hearts incubated under these conditions lost about one-half of their weight and protein during 2 days of incubation (data not shown) in accord with previous studies (10, 29). We determined whether the retardation of protein degradation by leupeptin could influence this loss of muscle protein. After 48 h of incubation with leupeptin, the total protein content of the hearts was 19% greater than that of untreated hearts (Table 1). In contrast, hearts treated with antipain, chymostatin, and pepstatin did not contain more protein than control hearts after 48 h in vitro (data not shown).
/
Lwpeptin
tY
I 48
HOURS FIG. 1. Tyrosine in incubation medium of cultured fetal mouse hearts was measured at times indicated on ordinate. A bcissa represents cumulative tyrosine release, a measure of net protein breakdown. Leupeptin decreased net protein breakdown at all concentrations studied (P < 0.01). Rate of net proteolysis in each group was different from all other groups (P c 0.05). Vertical bars indicate SEM.
Downloaded from www.physiology.org/journal/ajpendo at Macquarie Univ (137.111.162.020) on February 13, 2019.
LEUPEPTIN
DECREASES
PROTEOLYSIS
1. Effects of leupeptin fetal mouse hearts
AND
Net Protein Breakdown, nmol Tyr/Heart per 48 h
30 PM
ATROPHY
on cultured
TABLE
Control Leupeptin,
CARDIAC
3. Effects of leupeptin mouse hearts
TABLE
Protein:&;?
pg/
20.7 t, 0.4 10.2 AZ 0.2
NO*18 226 t 19
P < 0.001
P < 0.02
Values for net protein breakdown are means t SE pooled from at least 15 tyrosine determinations on incubation medium of at least 30 hearts from at least 7 litters studied at least 2 different times. For protein content, values are means k SE of 47 observations pooled from hearts studied at several different times. Proteins were determined after 48 h in vitro on the sodium hydroxide (0.5 M) digests of the whole hearts or of their perchloric’acid (0.2 M) insoluble material. The initial protein content was approximately twice that remaining after 48 h in the untreated hearts in accord with previous studies (2, 10).
2. Effects of protease inhibitors breakdown in fetal mouse hearts
on protein
TABLE
% Decrease Protein Breakdown
Antipain, 30 ,uM Chymostatin, 30 PM Pepstatin, 30 ,uM Soybean trypsin inhibitor, 100 pg/d Bovine lung protease inhibitor (Trasyw 100 M/d
19 12 10 4 4
P
LIBBY,
JOANNE
S. INGWALL,
AND
ALFRED
in
L. GOLDBERG of Medicine,
Department of Physiology, Harvard Medical School, and Department Peter Bent Brigham Hospital, Boston, Massachusetts 02115 LIBBY, PETER, JOANNE S. INGWALL, AND ALFRED L. GOLDBERG. Reduction of protein degradation and atrophy in cultured feta2 mouse hearts by Zeupeptin. Am. J. Physiol. 237(l): E35-E39,1979 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 6(l): E35-E39, 1979.-This study examined the effects of certain protease inhibitors on protein turnover, atrophy, and viability of cultured fetal mouse hearts. Leupeptin (30 PM) diminished net proteolysis by about 50% (P < 0.001) and as a consequence retarded cardiac atrophy. Hearts cultured with leupeptin for 2 days contained 19% more protein than control hearts (P < 0.02). Leupeptin did not alter microscopic appearance, pattern of contraction, rates of protein synthesis and protein leakage, or levels of adenosine triphosphate, lactate dehydrogenase, and creatine kinase. Other protease inhibitors (antipain, pepstatin, chymostatin, and those from soybean and bovine lung) had little or no effect on proteolysis and did not decrease atrophy. Leupeptin inhibits intracellular cathepsin B activity. However, in hearts exposed to leupeptin for 48 h and then washed to remove the inhibitor, cathepsin B level was twice control, whereas three other lysosomal hydrolase activities changed little or not at all. Thus, prolonged exposure to its inhibitor selectively increased tissue content of this lysosomal protease. These findings illustrate the importance of protein breakdown in determining tissue mass. Leupeptin appears useful in studies of protein turnover, in maintaining tissues or organs in culture, and possibly in the therapy of certain diseases. protein breakdown; cardiac muscle
cathepsin
B; protease
inhibitors;
lysosomes;
RATE of protein breakdown in a tissue is an important determinant of its protein content (6, 7,22, 31). Agents that specifically reduce this process could prove useful in investigations of the mechanism, control, and physiological role of intracellular protein breakdown. Because such agents should theoretically alter tissue growth or atrophy, they could also be useful in treatment of pathological states in which proteolysis is accelerated and in the maintenance of tissues and organs in culture. The protease inhibitor leupeptin decreases proteolysis in skeletal and atrial muscle (12) and isolated liver cells incubated in vitro for several hours (9, 11). Because cathepsin B activity is diminished in homogenates of leupeptin-treated muscles (12) and hepatocytes (unpublished observations), this lysosomal protease may be one site of the action of leupeptin. Previous reports have also suggested that mixtures of protease inhibitors containing leupeptin might be useful in preventing degeneration of muscles in animals with muscular dystrophy (15, 16, 24).
THE OVERALL
0363-6100/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
However the effective agent, its mechanism of action, and potential toxic effects of long-term exposure to protease inhibitors have not been investigated. Therefore this study investigated whether prolonged exposure to leupeptin reduces net proteolysis and protein loss from an atrophying tissue. These experiments also determined whether exposure to this agent for 2 days. produced toxic effects or altered the activities of lysosomal hydrolases. The fetal mouse heart offered several advantages for such a study. The hearts continue to beat for several days when incubated in defined media (10, 30). They atrophy markedly in vitro (10,30), perhaps because they do not contract against a load. Thus, this preparation permitted long-term observations under controlled conditions and allowed us to test the hypothesis that a protease inhibitor might selectively reduce protein loss from a differentiated tissue in negative nitrogen balance. MATERIALS
AND
METHODS
Chemicals and supplies. The protease inhibitors leupeptin, antipain, chymostatin, and pepstatin, peptide derivatives produced by actinomycetes (27), were kindly supplied by Prof. H. Umezawa (Tokyo, Japan). Bovine lung protease inhibitor (aprotonin, or Trasylol) was manufactured by Boehringer Mannheim Biochemicals (Indianapolis, IN). [3H]phenylalanine (7 mCi/mM) was purchased from Schwartz/Mann (Orangeburg, NY). Carbobenzoxy-alanyl-arginyl;arginyl-4-methoxy-2-naphthylamine and 4-methoxy-2-naphthylamine were supplied by Enzymes Systems Products (Livermore, CA). Soluene was purchased from Packard Instrument Co. (Downs Grove, IL). [ “C]methylglobin was prepared as previously described (20). Vitamins and antibiotics were bought from Grand Island Biochemical Co. (Grand Island, NY), and Falcon organ culture plates were bought from Becton-Dickenson & Co. (Oxnard, CA). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Organ Cultures. Hearts from fetuses of CD-l mice 1617 days postconception (Charles River Breeding Laboratories, Wilmington, MA) were incubated on a stainless steel screen at the medium/gas interface of organ culture plates, as previously described (29). Generally, two hearts were incubated per plate. The medium was Krebs-Ringer bicarbonate buffer, supplemented with glucose (10 mM) and insulin (0.1 U/ml) except as noted. All media were equilibrated with 95% 02: 5% CO2 and contained penicillin (100 U/ml), streptomycin 10 (pg/ml), and amphoterecin B 0.25 @g/ml). Society
Downloaded from www.physiology.org/journal/ajpendo at Macquarie Univ (137.111.162.020) on February 13, 2019.
E35
LIBBY,
E36
Net protein
degradation.
Net proteolysis, i.e., the balance between protein synthesis and degradation, was determined by measuring the release of tyrosine into the incubation medium (4, 5). Because this amino acid is neither synthesized nor degraded by cardiac muscle, its release into the medium reflects the net degradation of tissue proteins to amino acids (4, 5, 18)‘ The release of tyrosine underestimates the actual rate of protein breakdown because some of the tyrosine released by protein hydrolysis is reincorporated during protein synthesis. In these experiments we have ignored possible changes in the intracellular pools of tyrosine because these pools in cardiac muscle are much smaller than the amounts appearing in the medium in these long-term incubations (18)
Piotein synthesis and adenosine surements. After 48 h of incubation
triphosphate
mea-
in medium with or without leupeptin, hearts were incubated for 3 h with or without the inhibitor in Krebs-Ringer bicarbonate buffer supplemented with glucose (10 mM), insulin (0.1 U/ml), 5 times rat plasma concentrations of amino acids (15), except for phenylalanine (which was included at 0.5 mM final concentration), vitamins for minimal essential medium, and [3H]phenylalanine (15 &i/plate). Each heart was then immediately placed in cold perchloric acid (0.2 M), and homogenized, and 2 mg of bovine serum albumin (10% wt/vol in water) were added. After centrifugation, the pellet was washed twice in perchloric acid, once in ethanol-ethyl ether (1: 1 vol/vol), dissolved in Soluene (0.4 ml), and counted as described (6). Samples of the media were also counted. Efficiency of counting was determined by use of an automatic external standard. The rate of phenylalanine incorporation was calculated assuming that the specific activity of phenylalanine in the medium approximated that of the precursor pools for protein synthesis. This assumption is probably valid for hearts incubated with 0.5 mM unlabeled phenylalanine (17). The perchloric acid supernatant of these hearts was neutralized with potassium hydroxide and the volume noted. After centrifugation at 4°C to remove potassium perchlorate, the adenosine triphosphate concentration was determined in the supernatant photometrically using fnefly luciferase (23). Protein and enzyme assays. Protein was determined by the method of Lowry et al. (13) with bovine serum albumin as the standard. To measure protein released into the incubation medium, aliquots of media were pooled and treated with perchloric acid (final concentration 0.2 M) to precipitate proteins prior to assay. Cathepsin B was measured using the fluorogenic substrate carbobenzoxy-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine (12, 25), after washing the hearts to remove the inhibitor (Table 4). N-acetyl-P-glucosaminidase and acid phosphatase were measured calorimetrically by standard techniques (2). The leupeptin-insensitive acidic proteolytic activity was measured by incubating [‘“Clmethylglobin in sodium acetate buffer (0.1 M, pH 4) in the presence of leupeptin (3 PM). The release of radioactivity soluble in perchloric acid (0.2 M) was measured. Lactate dehydrogenase (3) and creatine kinase (21) were measured spectrophotometrically. All statistical calculations used Student’s t test for independent observations.
INGWALL,
AND
GOLDBERG
RESULTS
The fetal mouse hearts were in marked negative nitrogen balance. Net protein degradation occurred at a constant rate during 48 h of incubation, as indicated by the linear release of tyrosine from the tissue (Fig. 1, Table 1). At all concentrations tested, leupeptin reduced the rate of net protein breakdown (Fig. 1, Table 1). This agent decreased net protein breakdown to a similar extent in hearts incubated in medium supplemented with glucose alone, and those incubated in medium further enriched with insulin (0.1 U/ml) and 5 times normal rat plasma levels of amino acids (data not shown). Because mixtures of protease inhibitors have been reported to delay degeneration of dystrophic skeletal muscle (15, 16, 24), we also tested whether a variety of additional protease inhibitors also altered net protein breakdown. The compounds tested were antipain, pepstatin, and chymostatin, which are other peptide aldehydes produced by actinomycetes (27), and the polypeptide soybean trypsin inhibitor and bovine lung protease inhibitor (Trasylol or aprotonin). Of these compounds only antipain decreased net proteolysis in the cultured hearts, but its effect was much smaller than that of leupeptin (Table 2). The fetal mouse hearts incubated under these conditions lost about one-half of their weight and protein during 2 days of incubation (data not shown) in accord with previous studies (10, 29). We determined whether the retardation of protein degradation by leupeptin could influence this loss of muscle protein. After 48 h of incubation with leupeptin, the total protein content of the hearts was 19% greater than that of untreated hearts (Table 1). In contrast, hearts treated with antipain, chymostatin, and pepstatin did not contain more protein than control hearts after 48 h in vitro (data not shown).
/
Lwpeptin
tY
I 48
HOURS FIG. 1. Tyrosine in incubation medium of cultured fetal mouse hearts was measured at times indicated on ordinate. A bcissa represents cumulative tyrosine release, a measure of net protein breakdown. Leupeptin decreased net protein breakdown at all concentrations studied (P < 0.01). Rate of net proteolysis in each group was different from all other groups (P c 0.05). Vertical bars indicate SEM.
Downloaded from www.physiology.org/journal/ajpendo at Macquarie Univ (137.111.162.020) on February 13, 2019.
LEUPEPTIN
DECREASES
PROTEOLYSIS
1. Effects of leupeptin fetal mouse hearts
AND
Net Protein Breakdown, nmol Tyr/Heart per 48 h
30 PM
ATROPHY
on cultured
TABLE
Control Leupeptin,
CARDIAC
3. Effects of leupeptin mouse hearts
TABLE
Protein:&;?
pg/
20.7 t, 0.4 10.2 AZ 0.2
NO*18 226 t 19
P < 0.001
P < 0.02
Values for net protein breakdown are means t SE pooled from at least 15 tyrosine determinations on incubation medium of at least 30 hearts from at least 7 litters studied at least 2 different times. For protein content, values are means k SE of 47 observations pooled from hearts studied at several different times. Proteins were determined after 48 h in vitro on the sodium hydroxide (0.5 M) digests of the whole hearts or of their perchloric’acid (0.2 M) insoluble material. The initial protein content was approximately twice that remaining after 48 h in the untreated hearts in accord with previous studies (2, 10).
2. Effects of protease inhibitors breakdown in fetal mouse hearts
on protein
TABLE
% Decrease Protein Breakdown
Antipain, 30 ,uM Chymostatin, 30 PM Pepstatin, 30 ,uM Soybean trypsin inhibitor, 100 pg/d Bovine lung protease inhibitor (Trasyw 100 M/d
19 12 10 4 4
P
