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Biomedical subjects

A M Samarel

Publications and source records attributed to A M Samarel.

At least 73 records · Page 4Linked to original sources
PubMed1989

Effects of cysteine protease inhibitors on rabbit cathepsin D maturation.

To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed (24 h) to media containing leupeptin (0-10 mM), E 64 (0-10 mM), or chloroquine (0-50 microM). Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D (Mr 53,000) within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D (Mr 48,000). Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate (not detectable in control fibroblasts). Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation.

Animals↗
PubMed1989

Regional differences in the in vivo synthesis and degradation of myosin subunits in rabbit ventricular myocardium.

To test for regional differences in the rates of synthesis and degradation of contractile proteins during normal physiological growth of the heart in vivo, fractional rates of protein accumulation and synthesis were assessed for total protein, myosin heavy chain, myosin light chain, phosphorylatable myosin light chain, and actin in the right and left ventricular free walls of growing, New Zealand White rabbits. Fractional rates of protein accumulation were determined from regional protein content growth curves of total and individual myofibrillar proteins measured in 82 animals ranging from 1 day to 9 weeks of age. In vivo right and left ventricular fractional rates of protein synthesis were determined by the [3H]leucine constant infusion method in 9-week-old rabbits. At this age, right and left ventricular fractional accumulation rates for total protein, myosin subunits, and actin were found to be identical, thus allowing for the comparison of the effect of regional hemodynamic load on protein degradation independent of its effect on growth. Total protein and individual myofibrillar protein fractional degradative rates were then derived as the difference between fractional rates of synthesis and accumulation. The results indicate increased fractional synthesis and degradation of myosin heavy chain and light chains, but not actin, in the left compared to the right ventricular free wall. Accelerated left ventricular replacement of myosin subunits may be related to regional differences in hemodynamic load. These results help explain the apparent increase in the in vivo rate of myocardial protein degradation observed in several experimental models of ventricular hypertrophy.

Animals↗
PubMed1988

Attachment and maintenance of adult rabbit cardiac myocytes in primary cell culture.

The present observations demonstrate that quiescent calcium-tolerant adult rabbit cardiac myocytes can be isolated by collagenase-hyaluronidase perfusion and maintained in primary culture for at least 2 wk. Culturing large numbers of myocytes requires that the freshly isolated cells be attached to a suitable substratum such as laminin, type IV collagen, or fetal bovine serum. The cultured myocytes retain their rod-like morphology for approximately 7 days before gradually spreading into a flattened conformation by 14 days. During the 1st wk of culture, contaminating interstitial cells rapidly proliferate, making cultures unsuitable for long-term study. Pure myocyte populations can be established and maintained if freshly isolated cells are cultured in the presence of cytosine arabinoside (Ara-C, 10 microM). This antimetabolite does not appear to adversely affect high-energy phosphates, since ATP and creatine phosphate (CrP) content of the myocytes is maintained at levels normally found in biopsy samples of rabbit myocardium. These results illustrate that an energetically stable population of adult cardiac myocytes can be maintained in primary culture in sufficient numbers to make them useful for future investigations of myocyte function.

Adenosine Triphosphate↗
PubMed1988

Nonrandom turnover of actin and tubulin in cultured rabbit cardiac fibroblasts.

Total protein fractional rates of growth, synthesis, and degradation were assessed in primary cultures of rabbit cardiac fibroblasts. Differences in fractional growth rates were produced by subculturing cells at low density and growing them to confluence. Total protein fractional degradative rates were then derived by subtracting fractional growth rates from measured fractional synthetic rates (obtained in [3H]leucine pulse-labeling experiments). Actin and tubulin degradation were studied in similar rapidly and slowly growing cultures. [35S]methionine pulse-chase experiments, followed by dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, and densitometry were used to determine the amount of labeled actin and tubulin remaining in cultures at various times during the chase (0-96 h). The indirect study showed a substantially lower total protein fractional degradative rate during rapid vs. slow growth (0.04 +/- 0.13 vs. 0.42 +/- 0.01 d-1 at 2 and 15 days after subculture, respectively; P less than 0.01). At both growth rates, the disappearance of labeled actin and tubulin was delayed, suggesting a more complex model for their degradation than random decay. Serum deprivation of slowly growing fibroblasts increased the rate of disappearance of both proteins by eliminating the delay in their breakdown. Thus the suppression of protein degradation during rapid growth appears to result from the presence of relatively greater amounts of "new" actin and tubulin (and possible other long-lived proteins) that are kinetically distinct from the total intracellular pools of these proteins with respect to their susceptibility to proteolysis.

Actins↗
PubMed1987

Quantitative analysis of myofibrillar protein subunits: demonstration of large molar excesses of myosin light chains in rabbit ventricular myocardium.

We describe a method for the complete solubilization and quantitative analysis of individual myofibrillar proteins in whole tissue homogenates of ventricular myocardium using gradient dodecyl sulfate polyacrylamide gel electrophoresis and staining with 125I-labeled Coomassie brilliant blue. The procedure allows for the simultaneous quantification of myosin heavy chain, myosin light chain, phosphorylatable myosin light chain and actin from as little as 50 mg of tissue. Within-assay and between-assay variations range from 8.0% to 12.6% for each protein subunit. The method was applied to the determination of the subunit stoichiometry of purified myosin, and to the measurement of myosin and actin concentrations in the neonatal and adult rabbit heart. Furthermore, we provide quantitative biochemical evidence for the existence of large molar excesses of myosin light chains in tissue homogenates of both neonatal and adult rabbit ventricular myocardium.

Actins↗
PubMed1987

Protein synthesis and degradation during starvation-induced cardiac atrophy in rabbits.

To determine the relative importance of protein degradation in the development of starvation-induced cardiac atrophy, in vivo fractional synthetic rates of total cardiac protein, myosin heavy chain, actin, light chain 1, and light chain 2 were measured in fed and fasted rabbits by continuous infusion of [3H] leucine. In addition, the rate of left ventricular protein accumulation and loss were assessed in weight-matched control and fasted rabbits. Rates of total cardiac protein degradation were then estimated as the difference between rates of synthesis and growth. Fasting produced left ventricular atrophy by decreasing the rate of left ventricular protein synthesis (34.8 +/- 1.4, 27.3 +/- 3.0, and 19.3 +/- 1.2 mg/day of left ventricular protein synthesized for 0-, 3-, and 7-day fasted rabbits, respectively). Inhibition of contractile protein synthesis was evident by significant reductions in the fractional synthetic rates of all myofibrillar protein subunits. Although fractional rates of protein degradation increased significantly within 7 days of fasting, actual amounts of left ventricular protein degraded per day were unaffected. Thus, prolonged fasting profoundly inhibits the synthesis of new cardiac protein, including the major protein constituents of the myofibril. Both this inhibition in new protein synthesis as well as a smaller but significant reduction in the average half-lives of cardiac proteins are responsible for atrophy of the heart in response to fasting.

Animals↗
PubMed1986

Cardiac protein synthesis and degradation during thyroxine-induced left ventricular hypertrophy.

Assessment of cardiac protein metabolism in thyroxine-induced left ventricular hypertrophy requires measurements of both protein synthesis and degradation. In vivo protein degradative rates can best be measured as the difference between rates of protein synthesis and growth. Accordingly, rates of left ventricular protein accumulation were determined in growing rabbits, and in animals administered intravenous L-thyroxine (200 micrograms X kg-1 X day-1) for up to 15 days. Left ventricular protein fractional synthetic rates in euthyroid and thyroxine-treated rabbits were measured by continuous infusion of [3H]leucine (200 mu Ci/h X 6 h), and results converted to milligrams protein synthesized and degraded per day. Thyroxine administration produced left ventricular hypertrophy by increasing the rate of total protein synthesis (35.7 +/- 2.0, 71.0 +/- 7.0, and 62.6 +/- 4.0 mg of left ventricular protein synthesized per day for 0-, 3-, and 9-day, thyroxine-treated rabbits, respectively). However, the increased rate of total protein synthesis was greater than the measured rate of total protein accumulation (8.1 vs. 15.9 mg protein/day for euthyroid and thyroxine-treated animals), indicating that left ventricular protein degradative rates were increased as well. These studies indicate that accelerated proteolysis may be important in the molecular and architectural remodeling of the rapidly hypertrophying heart during thyrotoxicosis.

Animals↗
PubMed1986

Lysosomal changes during thyroxine-induced left ventricular hypertrophy in rabbits.

By use of a combined morphologic, immunocytochemical, and biochemical approach, this study demonstrates the changes in the lysosomal vacuolar apparatus that accompany thyroxine-induced cardiac hypertrophy. During the 1st wk of thyroxine administration, immunocytochemical studies revealed a decline in cathepsin D within many myocytes, but an increase within interstitial cells. These events transpired with only a modest rise in cathepsin D activity. During the 2nd wk, cathepsin D reappeared within most myocytes and continued to increase within the interstitial population and was associated with a general rise in lysosomal enzyme activity. These data demonstrate the importance of employing both immunocytochemical and biochemical approaches to evaluate the status of the lysosomal vacuolar apparatus, and suggests that the lysosomal vacuolar apparatus may be involved in the apparent remodeling that attends rapid cardiac growth, which may represent one component of the enhanced rates of proteolysis documented in this model of cardiac hypertrophy.

Animals↗
PubMed1986

Release of unassembled rat cardiac myosin light chain 1 following the calcium paradox.

To determine the intracellular source and release kinetics of myosin light chain 1 immediately following irreversible myocytic injury, we perfused rat hearts in a Langendorff apparatus under control conditions (20 minutes), or during global cellular injury produced by oxygenated, calcium-free perfusion (5 minutes), followed by reperfusion with buffer containing 2.5 mM calcium (15 minutes). Light chain 1 concentration (double antibody radioimmunoassay) and creatine kinase activity were measured in both the coronary effluent and the 140,000 g supernatant extract of perfused ventricular tissue (after homogenization and ultracentrifugation). Calcium reperfusion caused the rapid release of both light chain 1 and creatine kinase activity (peak light chain 1 = 1.09 +/- 0.19 micrograms/g; peak creatine kinase = 74.9 +/- 10.7 IU/ g at 1 minute, mean +/- SD, n = 3); 28.5 +/- 13.5% of total light chain 1 and 86.5 +/- 0.6% of total creatine kinase activity were depleted from the tissue extract during the 15-minute reperfusion. No light chain 1 or creatine kinase was detected in the effluents of control-perfused hearts. Dodecyl sulfate polyacrylamide gel electrophoresis and immunodetection with specific antibody to myosin heavy chain and light chain 1 showed that the effluent light chain 1 was of similar molecular weight (mol wt = 27,000) to the subunit bound to myofibrils. In addition, light chain 1 was released in the absence of myosin heavy chain. Thus, a small soluble pool of unassembled myosin light chain 1 subunits exists in the cytoplasm of cardiac myocytes that is released from irreversibly injured cells. This pool demonstrates initial washout kinetics similar to creatine kinase.

Animals↗
PubMed1985

Identification of artifactual cathepsin D activity in cardiac subcellular fractions related to formation of an iron-EDTA complex.

The conventional assay procedure for cathepsin D (E.C.3.4.23.5) activity in tissue homogenates and subcellular fractions requires incubation with hemoglobin as substrate. Cathepsin D (CD) activity is calculated by determining the increase in absorbance at 280 nm after precipitation of all proteins with trichloroacetic acid. This increase in absorbance (presumably due to the release of tyrosine residues from hemoglobin) is converted to arbitrary CD activity units. Homogenization and fractionation of cardiac tissue frequently requires that ethylenediamine tetraacetic acid (EDTA) be included in the homogenization medium. We have observed that subcellular fractions of cardiac tissue prepared in the presence of EDTA demonstrate residual CD activity despite either quantitative removal of all CD protein by immunoprecipitation or complete inhibition of CD by pepstatin. The present study demonstrates that this 'apparent' CD activity (residual increase in absorbance at 280 nm) is due to the formation of an Fe-EDTA complex which absorbs at 280 nm. Data are presented which demonstrates that the EDTA of the medium complexes with non-heme iron which contaminates commercially available hemoglobin. A method for preparing hemoglobin free of contaminant non-heme iron is described for use in studies of CD metabolism when EDTA is present in the homogenization buffer.

Animals↗
PubMed1985

Transport and proteolytic processing of rabbit cardiac cathepsin D.

Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.

Animals↗
PubMed1984

Biosynthesis of the multiple forms of rabbit cardiac cathepsin D.

Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.

Animals↗
PubMed1983

Susceptibilities of cardiac myofibrillar proteins to cathepsin D-catalyzed degradation.

The binding of various myofibrillar proteins to cathepsin D and consequent susceptibility to degradation was assessed by determination of apparent Michaelis constant (Km) values for the purified enzyme using myosin, alpha-actinin, actin, and tropomyosin as substrates. Cathepsin D, purified 1,000-fold to homogeneity from canine cardiac tissue, was incubated (0-100 min at 37 degrees C) with myofibrillar proteins isolated from homologous tissue and labeled radioactively with 14C by reductive alkylation. The reaction was terminated by addition of trichloroacetic acid (10% wt/vol), and radioactivity in the supernatant fraction was determined after centrifugation (5 min) at 100,000 g. Double reciprocal graphs (1/reaction velocity vs. 1/substrate concn) were constructed for each substrate from the linear portion of graphs of disintegrations. min-1 X ml-1 100,000-g supernatant vs. time (min). Apparent Km values (+/- SD) calculated for myosin, alpha-actinin, actin, and tropomyosin were found to be 2.7 +/- 0.3 X 10(-6) (n = 6), 10.0 +/- 2.3 X 10(-6) (n = 13), 13.0 +/- 1.3 X 10(-6) (n = 7), and 45.5 +/- 9.0 X 10(-6) (n = 6) mol/l, respectively. The results show that myofibrillar proteins differ in their binding and thus susceptibility to hydrolysis by cathepsin D in order of molecular weight (i.e., myosin greater than alpha-actinin greater than actin greater than tropomyosin). Because the relative turnover rates of myofibrillar proteins are known to be independent of molecular weight, our results suggest that the degradation of these proteins by cathepsin D is not rate limiting and that other factors including lysosomal or nonlysosomal enzymes must determine the rate-limiting steps of myofibrillar protein degradation in cardiac tissue.

Actinin↗
PubMed1982

Regulation of cathepsin D metabolism in rabbit heart: evidence for a role for precursor processing in the control of enzyme activity.

Production of active lysosomal enzymes may involve limited proteolysis of inactive high molecular weight precursors. Precursor processing potentially regulates lysosomal enzyme activity. To test whether rabbit cardiac cathepsin D is first synthesized as a precursor and whether prolonged fasting (a condition affecting both cathepsin D and total cardiac protein turnover) influences precursor processing, rates of cathepsin D synthesis and processing were compared in left ventricular slices of control and 3-d-fasted rabbits incubated in vitro with [(35)S]methionine. (35)S-labeled cathepsin D was isolated by butanol-Triton X-100 extraction, immunoprecipitation, and dodecyl sulfate-polyacrylamide gel electrophoresis. Total cardiac protein synthesis was measured by tracer incorporation and normalized for differences in precursor pool size by direct measurement of [(35)S]aminoacyl-tRNA-specific radioactivity. Relative cathepsin D synthetic rates were obtained by comparing (35)S incorporation into cathepsin D with (35)S incorporation into all cardiac proteins. Enzyme processing was assessed in pulse-chase experiments and assayed by autoradiography. The results indicate that (a) rabbit cardiac cathepsin D is synthesized as a precursor (53,000 mol wt) that is processed to a 48,000-mol wt form, (b) rates of both cathepsin D and total cardiac protein synthesis are similar in control and fasted rabbits, suggesting that decreased enzyme degradation rather than increased synthesis is responsible for the elevated levels of cardiac cathepsin D in starvation, and (c) cathepsin D processing in hearts of fasted animals is incomplete, with accumulation of the precursor during pulse-chase experiments of 6 h duration. Based upon these results, a three-stage model for the regulation of cathepsin D activity in rabbit heart is proposed.

Animals↗
PubMed1981

A double antibody radioimmunoassay for canine cardiac cathepsin D.

We have developed a sensitive double antibody radioimmunoassay for measuring canine cardiac cathepsin D. Radioiodinated cathepsin D was prepared by chloramine T oxidation using a highly purified source of enzyme. High avidity antiserum to the canine cardiac enzyme was raised in rabbits. Antibody-bound cathepsin D was separated from free enzyme using goat anti-rabbit IgG second antibody. The least amount of immunoreactive enzyme measurable in the radioimmunoassay was 2.4 ng.cm-3 as determined by antibody titration. The assay was linear for concentrations of enzyme in the range of 10 to 120 ng.cm-3. Within-assay and between-assay variations were 12%. The radioimmunoassay described was used to measure the immunoreactive cathepsin D content of the 100 000 x g supernatant fraction of canine myocardial homogenates.

Animals↗
PubMed1981

Rabbit cardiac immunoreactive cathepsin D content during starvation-induced atrophy.

To determine whether the increased activity of cathepsin D observed during starvation-induced cardiac atrophy results from activation of preexisting enzyme or synthesis of new enzyme, a solid phase double-antibody radioimmunoassay was developed for measurement of immunoreactive cathepsin D in extracts of rabbit myocardium. Cathepsin D activity was significantly increased in the hearts of animals starved for 3, 7, and 14 days (82.6 +/- 0.8, 87.2 +/- 3.8, and 95.3 +/- 3.5 U/g wet wt, respectively) compared to controls (65.5 +/- 1.4 U/g wet wt; P less than 0.001). Immunoreactive cathepsin D was increased to a greater extent (168 +/- 7, 179 +/- 16, 200 +/- 17, and 104 +/- 5 micrograms/g wet wt for 3-, 7-, and 14-day starvation and controls, respectively; P less than 0.001) than that expected on the basis of the observed increase in enzyme activity. Sephadex G100 gel filtration of cardiac lysosomal extracts from starved and control animals revealed no evidence of high or low molecular weight forms of cathepsin D. The results suggest the observed increase in cathepsin D activity during starvation-induced cardiac atrophy is accompanied by an increased synthesis and/or decreased degradation of cathepsin D protein, rather than activation of preexisting enzyme. The lower activity levels observed during starvation possibly result from alterations in the concentrations of endogenous inhibitors or activators of cathepsin D.

Acid Phosphatase↗