Wieland Gevers

Increased cardiac myosin ATPase activity as a biochemical adaptation to running training: Enhanced response to catecholamines and a role for myosin phosphorylation

Abstract The effects were studied of prior running training on protein phosphorylation and adenosine triphosphatase (ATPase) activities of natural actomyosin isolated from perfused rat hearts. Myosin Ca 2+ -ATPase activities were significantly higher in running-trained hearts than in controls, whereas the Ca 2+ -stimulated, Mg 2+ -dependent ATPase activities of natural actomyosin were not changed. After treatment of isolated perfused hearts with the β-agonist isoproterenol, both troponin-I and myosin P light chains became phosphorylated. Troponin-I phosphorylation (1 mol/mol) was the same in both sets of hearts and was accompanied by similar changes in cardiac cyclic AMP contents. The V max values for myosin Ca 2+ -ATPase activity were increased after isoproterenol treatment in all the perfused hearts, but to a significantly greater extent in the hearts of running trained animals; this was correlated with enhancement of both the rate and extent of myosin P light chain phosphorylation. Enhanced Ca 2+ -dependent myosin P light chain phosphorylation, further enhanced by β-adrenergic stimulation, represents, at the molecular level, a biochemical response to running training.

Low density lipoprotein receptor mutations in South African homozygous familial hypercholesterolemic patients.

The binding and catabolism of low density lipoprotein (LDL) were studied in cultured skin fibroblasts and/or Epstein-Barr virus-transformed lymphoblastoid cells from 20 familial hypercholesterolemic (FH) homozygotes of 14 Afrikaner kindreds. Cells from available heterozygotes were also analyzed. Good resolution between the normal, heterozygote, and homozygote groups was obtained on the basis of these assays using either cell type. Results obtained with fibroblasts allowed the classification of 13 of these kindreds as being typically receptor-defective, and one kindred with two homozygotes as being receptor-negative. Fibroblasts from homozygotes of the receptor-defective class expressed LDL receptor activities which varied between 3% and 25% of normal. Where two homozygotes from the same family were available for assays (six families), both yielded similar activities. In contrast to fibroblasts, all the lymphoblastoid cells derived from FH homozygotes yielded LDL receptor activities equal to or less than 10% of normal; in a number of cases no significant 125I-LDL binding was detectable. The use of lymphoblastoid cells provides a convenient means for screening for FH at the cellular level. Our results indicate a predominance of a receptor-defective type of abnormality, which is consistent with a founder gene effect in the Afrikaner population.

Oxidative phosphorylation in infarcting baboon and dog myocardium: effects of mitochondrial isolation and incubation media.

Abstract Mitochondrial changes during ischaemia and infarction were studied in hearts from dogs and from Cape Chacma baboons after coronary artery ligation. At different time intervals after coronary artery ligation, tissue biopsies were taken from four areas: (1) the apparent centre of the infarcting area; (2) the periphery of the infarcting area; (3) apparently normal tissue adjacent to the infarction; and (4) left ventricular tissue far from the infarcting area. One hour after coronary artery ligation, the results obtained were dependent on the mitochondrial isolation and incubation media used. In the infarcting myocardium, phosphorylation/oxidation ratios (P/O ratios), determined manometrically or polarographically, were significantly depressed, as was the respiratory control index. P/O ratios were more significantly depressed in mitochondria isolated in sucrose than in KC1-EDTA-albumin (KEA). Using a fortified incubation medium and sucrose-EDTA or KEA as isolation medium, the mitochondrial oxygen uptake was similar in all areas studied, suggesting a specific defect in phosphorylation. Using the polarographic technique, with KEA as isolation medium and a relatively simple incubation medium, the oxygen uptake of mitochondria from infarcting tissue was depressed. After 6 h of ischaemia, mitochondrial oxygen uptake, phosphorylation and P/O ratios were all depressed even in a fortified medium.

Low density lipoprotein receptor founder mutations in Afrikaner familial hypercholesterolaemic patients: a comparison of two geographical areas.

SummaryAfrikaners with familial hypercholesterolaemia (FH) were screened for the presence of three point mutations in the low density lipoprotein receptor gene that were previously described as being relatively common in this population. The prevalence and distribution of the mutations were compared in 27 unrelated homozygous and 79 unrelated heterozygous FH Afrikaner patients from two regions in South Africa, the Transvaal and Cape Provinces. The relative distribution of the three mutations was similar in the two regions, with the FH1 mutation being the most prevalent (66%), followed by the FH2 mutation (27%) and the FH3 mutation (7%). Interestingly, defects other than the three common mutations are more common in the Cape than in the Transvaal; thus the three known mutations account for 98% of FH alleles in the Transvaal and only 74% in the Cape Province. None of the patients carried the recently described familial defective apolipoprotein B100 mutation. These results establish that three “founder” mutant genes occur amongst the Afrikaner and are responsible for the overall high prevalence of FH in this population.

A high-molecular-weight cysteine endopeptidase from rat skeletal muscle.

A cytosolic enzyme of high molecular weight (about 500 000), which attacks native or denatured proteins (inter alia, casein, globin and hexokinase) was purified about 1000-fold from mixed rat skeletal muscles, including muscles freed of mast cells by prior treatment of the animals with the degranulator, compound 48/80. Peptides of varying size were generated from radioactively labelled globin, but no free amino acids were formed; free tyrosine was also not released from azocasein. The pH optimum was 7.5 and the presence of an essential cysteine group was suggested because dithiothreitol (1 mM) stimulated the activity and N-ethylmaleimide (5 mM) and p-chloromercuriphenylsulphonic acid (1 mM) were inhibitors. The activity was markedly inhibited by Zn2+ but not by leupeptin, chymostatin or pepstatin. The enzyme was stabilized by ATP, at concentrations as low as 0.1 mM, against inactivation at 42 degrees C. The endopeptidase was clearly separated on gel chromatography from another large protease, also sensitive to Zn2+, but with marked aminopeptidase activity and the properties of hydrolase H. The activity levels of the protease, assayed after chromatography on Sepharose 6B of high-speed supernatant fractions, did not vary significantly in skeletal muscle samples which were derived from denervated, starved, diabetic or hyperthyroid animals, in all of which the abnormal physiological states expressed themselves as enhanced rates of tyrosine released by incubated soleus and extensor digitorum longus muscles. Nevertheless, the enzyme described here may be part of an ATP-dependent, multi-component proteolytic system similar to that already known to be present in reticulocytes.

Elastin biosynthesis by smooth muscle cells cultured under scorbutic conditions.

Abstract The proliferation of rat heart smooth muscle cells is unaffected by supplementation of the culture medium with ascorbic acid. The presence or absence of the vitamin has a pronounced effect, however, on the amount of alastin which is produced. Scorbutic cultures incorporate significantly more radioactive valine (an amino acid prevalent in elastin) into proteins present in the extracellular matrix than do supplemented controls, while there was no difference between the systems in the incorporation of labelled methionine (absent from elastin). Deficiency of ascorbic acid appears to result in an enhancement of the biosynthesis and extracellular processing of elastin in this culture system.

Low density lipoprotein metabolism in cultured fibroblasts from a new group of patients presenting clinically with homozygous familial hypercholesterolemia.

The metabolism of low density lipoproteins (LDL) was studied in cultured fibroblasts obtained from five local patients diagnosed, on the basis of clinical features and serum cholesterol concentrations, as having the homozygous form of familial hypercholesterolemia. LDL receptor function was assessed by measuring the binding, internalization, and degradation of 125I-labeled LDL, and by measuring the stimulation of cellular acyl-CoA cholesterol acyltransferase (ACAT) activity which followed exposure to LDL. Fibroblasts from two cases (CF and GM) showed receptor activities which were approximately 10% of the values obtained with normal cells, while ACAT stimulation by LDL was very low. These two patients were classified as homozygous for a receptor-defective abnormality. However, fibroblasts from the other three patients (JG, ES, and TT) showed greater than 25% of normal receptor activity, as assessed by 125I-LDL binding and catabolism. ACAT stimulation by LDL in cells from JG and ES was within the range of values shown by cells previously characterized as heterozygous for a receptor-negative mutation. Cells from ES behaved atypically: a low, but nonsaturable, activation by LDL was evident. ACAT stimulation by LDL was normal in cells from TT. Receptor activities of the cells from the available parents, assessed on the basis of LDL binding and degradation or of ACAT stimulation, were not clearly distinguishable from those of normal cells. These results add to the growing evidence of genetic heterogeneity underlying the clinical picture associated with familial hypercholesterolemia in different geographical distributions.

Effects of extracellular calcium concentrations on myosin P light chain phosphorylation in hearts from running-trained rats

Abstract Certain biochemical responses to clevated perfusate calcium concentrations were studied in the isolated hearts of sedentary and running-trained rats from which natural actomyosin was prepared. Both the Vmax and Ca2+ sensitivity of the Ca2+-stimulated, Mg2+-dependent actomyosin adenosine triphosphatase (ATPase) were similar in the hearts of trained and sedentary animals, and were not altered when the concentration of extracellular Ca2+ was increased. Enhanced myosin Ca2+-ATPase activities and phosphate contents of myosin P light chains were found in the hearts of trained animals compared with controls, and these differences were still maintained when perfusate Ca2+ concentrations were increased. Although treatment of the perfused hearts with isoproterenol also increased both parameters in the trained and sedentary series, the Vmax for myosin Ca2+-ATPase and the alkali-labile phosphate contents of myosin P light chains remained, throughout, significantly greater in the hearts of trained rats than in their sedentary counterparts. These differences were not eliminated by the combined use of isoproterenol and high perfusate Ca2+. The results suggest that an enhanced capacity for trans-sarcolemmal Ca2+ flux in the hearts of trained animals may be responsible for enhanced Ca2+-dependent phosphorylation of myosin P light chains, and thus improving cardiac function.

Altered adenosine triphosphatase activities of natural actomyosin from rat hearts perfused with isoprenaline and ouabain

Abstract Perfused rat hearts were treated with isoprenaline (10−6M) or ouabain (5.5 × 10−6M). The phosphate contents of troponin-I and myosin P light chains were established by radiolabelling with 32P; in the case of the light chains, direct chemical analysis of total and of specifically alkali-labile phosphate was also performed. Addition of isoprenaline caused phosphorylation of both troponin-I and myosin P light chains, reaching a maximum increment, after several minutes, of 1 mol/mol and 0.30 mol/mol, respectively. The Mg2+-ATPase activities, at saturating Ca2+ concentrations, of natural actomyosin isolated from treated hearts were significantly depressed, and an inverse correlation was established between the phosphate content of troponin-I and the Vmax[Ca2+] of this ATPase activity. The Ca2+ sensitivity of the Ca 2+ Mg 2+ -ATPase was also decreased. These changes were all reversed by an incubation permitting dephosphorylation of proteins by endogenous phosphatases. Treatment of hearts with ouabain caused no increment in troponin-I phosphorylation, but increased the P light chain phosphate content to a maximum of 0.30 mol/mol after some minutes. A positive correlation was evident between phosphate content of the light chains (in all experiments) and the maximum myosin Ca2+-ATPase activities. In addition, the Vmax[ATP] of the Ca 2+ Mg 2+ -ATPase of natural actomyosin was increased when light chain phosphorylation had occurred in the absence of troponin-I phosphorylation. P-light chain phosphorylation did not affect the Ca2+ sensitivity of Ca 2+ Mg 2+ -ATPase activity. We suggest that the effects of phosphorylation of troponin-I are to diminish thin filament sensitivity to Ca2+, and to decrease the efficiency of the transduction process along neighbouring actin monomers, such that the number of actin-myosin crossbridge interactions is decreased even in the presence of Ca2+ excess. Phosphorylation of P light chains of myosin has an activating effect on myosin Ca2+-ATPase activity, as well as on the rate of cross-bridge formation.

The surface distribution of low density lipoprotein receptors on cultured fibroblasts and endothelial cells

SummaryThe distribution of low density lipoprotein (LDL) receptors marked with colloidal gold-conjugated low density lipoproteins has been mapped on the surfaces of cultured human skin fibroblasts and bovine aortic endothelial cells viewed whole in the transmission electron microscope. A dispersed or scattered population of LDL receptors, in addition to and clearly distinct from clustered receptors was detected on the surfaces of both fibroblasts and dividing endothelial cells. No LDL receptors could be detected on contact-inhibited endothelial cells. Clustered receptors imaged in whole-mount preparations were often arranged in rings with an approximate diameter of 250 nm. In ultra-thin sections of marked cells, clustered receptors were localised in coated pits while the few dispersed receptors seen were restricted to non-coated membrane regions. Clustered receptors often appeared localised on the rims of coated pits whose central areas were not marked. The dispersed population of receptors was usually distributed diffusely amongst the clusters on dividing endothelial cells and normal fibroblasts. Only the dispersed population appeared on LDL receptor internalisation-defective mutant fibroblasts. The marginal zones of both fibroblasts and dividing endothelial cells were populated by dispersed receptors. Clusters appeared further “inland” and were rarely seen near the cell margins. These results indicate that LDL receptors on dividing endothelial cells and fibroblasts may be dispersed on the cell surface upon or soon after their insertion during recycling.