Evidence for increased synthesis as well as increased degradation of protein kinase C after treatment of human osteosarcoma cells with phorbol ester

Phorbol 12-myristate 13-acetate (PMA) induces time-dependent changes in protein kinase C subcellular distribution and enzymatic activity in the human osteosarcoma cell line SaOS-2. Short (less than 60 min) incubations with PMA caused decreased cytosolic enzyme activity and a concomitant increase in particulate protein kinase; after 3 h, particulate protein kinase C activity also declined to reach less than 10% of basal activity by 24 h (Krug, E., and Tashjian, Jr., A. H., (1987) Cancer Res. 47, 2243-2246). In order to determine whether the loss in enzyme activity was due to decreased enzyme protein, Western blot analyses were performed using a polyclonal antibody against protein kinase C raised in rabbits. This approach confirmed the previously reported time-related changes: 80-kDa immunoreactive protein kinase C initially translocated from the cytosol to the particulate cell fraction and later disappeared completely from the particulate fraction. Loss of protein kinase C enzymatic activity thus results from actual loss of the 80-kDa protein; we found no evidence for generation of a calcium/phospholipid-independent protein kinase C-like form of the enzyme. Membrane association was confirmed by immunoprecipitation experiments using [35S]methionine-labeled cells. Brief exposure to PMA caused a marked loss in the [35S]methionine-labeled cytosolic protein kinase C band and an increase in the labeled particulate band. Protein kinase C immunoprecipitated from cells treated with PMA for 14 h displayed an increase in [35S]methionine label despite a greater than 80% loss of enzyme activity. The high specific radioactivity of the remaining 80-kDa protein leads us to conclude that long term treatment with PMA causes an increase in the rate of protein kinase C synthesis accompanied by a still greater increase in the rate of enzyme degradation in SaOS-2 cells.     

Evidence for a regulatory action of vanadate on protein phosphorylation in brain microvessels

We investigated the action of vanadate on protein phosphorylation in microvessels isolated from rat brain. We found that a stimulation of protein phosphorylation from 32P-ATP occurs, in the presence of different concentrations of vanadate, 10(-3) M being the most effective dose. This action was time-dependent, and it was more evident after 60 s of treatment. The contribution of ATPase inhibition caused by vanadate appears to be negligible. In addition a stimulation of cAMP-dependent protein kinase activity was observed. The pattern of protein phosphorylation showed that exposure to 10(-3) M vanadate resulted in a nonspecific stimulation of protein phosphorylation concomitantly with a selective inhibition of the 55 KDa protein phosphorylation. The nature of this protein is also discussed.     

Factors involved in increased protein synthesis in rat kidney after administration of DDT

Homogenates of kidney cortex obtained from control rats and rats treated with DDT have been separated into microsomes or ribosomes, and into postmicrosomal (S105) supernatant fraction or pH 5 supernatant fraction. The incorporation of [¹⁴C]phenylalanyl-tRNA into peptide was increased when microsomes derived from kidneys of DDT-treated rats were incubated with pH 5 supernatant fraction from control rats. Elongation factors (EF) 1 and 2, necessary for the binding of aminoacyltRNA to ribosomes and for translocation of peptidyl-tRNA from the A site to the P site of ribosomes, were present in the pH 5 supernatant fractions of kidney of control and DDT-treated rats and these fractions were incubated with KCl-washed ribosomes obtained from livers of control rats. The results provided evidence that the increased incorporation observed with the pH 5 supernatant fraction obtained from the DDT-treated animals could not be attributed to decreased ribonuclease activity or to increased elongation factor 2 activity but was due to an increase in elongation factor l activity.     

Effects of chronic alcohol consumption on regulation of myocardial protein synthesis

Heart disease represents an important etiology of mortality in chronic alcoholics. The purpose of the present study was to examine potential mechanisms for the inhibitory effect of chronic alcohol exposure (16 wk) on the regulation of myocardial protein metabolism. Chronic alcohol feeding resulted in a lower heart weight and 25% loss of cardiac protein per heart compared with pair-fed controls. The loss of protein mass resulted in part from a diminished (30%) rate of protein synthesis. Ethanol exerted its inhibition of protein synthesis through diminished translational efficiency rather than lower RNA content. Chronic ethanol administration decreased the abundance of eukaryotic initiation factor (eIF)4G associated with eIF4E in the myocardium by 36% and increased the abundance of the translation response protein (4E-BP1) associated with eIF4E. In addition, chronic alcohol feeding significantly reduced the extent of p70S6 kinase (p70(S6K)) phosphorylation. The decreases in the phosphorylation of 4E-BP1 and p70(S6K) did not result from a reduced abundance of mammalian target of rapamycin (mTOR). These data suggest that a chronic alcohol-induced impairment in myocardial protein synthesis results in part from inhibition in peptide chain initiation secondary to marked changes in eIF4E availability and p70(S6K) phosphorylation.     

Impact of increased infarct transmurality on remodeling and function during healing after anterior myocardial infarction in the dog.

To determine the impact of greater infarct transmurality on changes in left ventricular remodeling and function after acute anterior myocardial infarction, serial topographic and functional parameters (two-dimensional echocardiograms) and hemodynamics over 6 weeks, and postmortem topography (planimetry) at 6 weeks, were measured in chronically instrumented dogs randomized to standard coronary artery ligation (group 1) or a modified lower ligation plus collateral obliteration to decrease collateral inflow and increase transmurality (group 2). At 6 weeks, postmortem scar size and collagen were similar in the two groups, but group 2 had greater transmurality associated with more necrosis relative to area at risk, Q waves, infarct expansion, thinning, regional bulging, and cavity dilatation. Over the 6 weeks, group 2 showed more early expansion, late thinning and regional bulging in the short axis, larger diastolic and systolic volumes, and more apical aneurysmal bulging in the long-axis, reflecting more topographic deterioration. More important, group 2 showed greater regional and global left ventricular dysfunction over 6 weeks, lower ejection fraction at 2 days with further decrease over 6 weeks, and more left ventricular thrombus, ventricular arrhythmias, and deaths. In addition, transmurality correlated with the severity of remodeling and dysfunction. The findings indicate that transmurality is a major determinant of remodeling and left ventricular dysfunction during healing after anterior infarction.     

Evidence for active control of perfusion within lung microvessels

Vasoconstrictors cause contraction of pulmonary microvascular endothelial cells in culture. We wondered if this meant that contraction of these cells in situ caused active control of microvascular perfusion. If true, it would mean that pulmonary microvessels were not simply passive tubes and that control of pulmonary microvascular perfusion was not mainly due to the contraction and dilation of arterioles. To test this idea, we vasoconstricted isolated perfused rat lungs with angiotensin II, bradykinin, serotonin, or U46619 (a thromboxane analog) at concentrations that produced equal flows. We also perfused matched-flow controls. We then infused a bolus of 3 μm diameter particles into each lung and measured the rate of appearance of the particles in the venous effluent. We also measured microscopic trapping patterns of particles retained within each lung. Thirty seconds after particle infusion, venous particle concentrations were significantly lower (P ≤ 0.05) for lungs perfused with angiotensin II or bradykinin than for those perfused with U46619, but not significantly different from serotonin perfused lungs or matched flow controls. Microscopic clustering of particles retained within the lungs was significantly greater (P ≤ 0.05) for lungs perfused with angiotensin II, bradykinin, or serotonin, than for lungs perfused with U46619 or for matched flow controls. Our results suggest that these agents did not produce vasoconstriction by a common mechanism and support the idea that pulmonary microvessels possess a level of active control and are not simply passive exchange vessels.     

Inflating bacterial cells by increased protein synthesis

Understanding how the homeostasis of cellular size and composition is accomplished by different organisms is an outstanding challenge in biology. For exponentially growing Escherichia coli cells, it is long known that the size of cells exhibits a strong positive relation with their growth rates in different nutrient conditions. Here, we characterized cell sizes in a set of orthogonal growth limitations. We report that cell size and mass exhibit positive or negative dependences with growth rate depending on the growth limitation applied. In particular, synthesizing large amounts of “useless” proteins led to an inversion of the canonical, positive relation, with slow growing cells enlarged 7‐ to 8‐fold compared to cells growing at similar rates under nutrient limitation. Strikingly, this increase in cell size was accompanied by a 3‐ to 4‐fold increase in cellular DNA content at slow growth, reaching up to an amount equivalent to ~8 chromosomes per cell. Despite drastic changes in cell mass and macromolecular composition, cellular dry mass density remained constant. Our findings reveal an important role of protein synthesis in cell division control.     

Delayed development of hypertension and increased aortic relaxation in spontaneously hypertensive rats after neonatal sympathectomy

The effect of neonatal sympathectomy on vasodilator responses to acetylcholine (ACh) and cAMP has been studied in aortic rings of spontaneously hypertensive rats (SHR) and normotensive animals. The relaxation of intact SHR aorta in response to ACh and cAMP was 20-35% lower than that of normotensive rats. Sympathectomy in normotensive rats did not affect the level of blood pressure and aorta reactivity to Ach. In SHR, sympathectomy caused a decrease in blood pressure, while relaxation in response to ACh and cAMP increased, as compared to intact SHR, but remained lower than in normotensive rats. The data obtained suggest that the decrease in arterial pressure of sympathectomized SHR is a result not only of the reduction in sympathetic effects but also of the increase in smooth muscle relaxation.     

Evidence for control of protein synthesis in HeLa cells via the elongation rate

The effects of fresh medium and serum on protein synthesis in suspension-cultured HeLa cells after growth to high cell density (>5 × 10⁵ cells/ml) were studied. Cells which were resuspended in fresh medium plus serum and grown for 24 hours (control) were compared with cells grown for 2 hours after resuspension (stimulated). The spectrum of proteins being synthesized by control and stimulated cells does not appear to be grossly different; that is, the weight and number average molecular weights of newly synthesized whole-cell protein are about the same in both cultures. Also, no significant differences were observed in the number of ribosomes per polysome or in the fraction of total ribosomes in polysomes. However, the transit times (combined elongation and termination times) were found to differ significantly; the average transit time for control cells was 2.24 minutes, while the average transit time for stimulated cells was 1.26 minutes. (An appendex evaluating the methodology involved in measuring the transit time is included.) In agreement with the difference in transit time, the absolute rate of protein synthesis in stimulated cells was approximately 1.8 times the rate measured in control cells. These data are taken as evidence that under certain conditions, the rate of elongtion and/or termination of polypeptide chains limits the overall rate of translation, and that cells can respond to growth conditions by changing the elongation and/or termination rate of protein synthesis.     

Evidence for enhanced eNOS function in coronary microvessels during the second window of protection

Nitric oxide (NO) derived from endothelial NO synthase (NOS) (eNOS) has been identified as a trigger for the second window of protection (SWOP), but its role as a mediator during the SWOP is a matter of debate. Eighteen mongrel dogs were chronically instrumented to measure left ventricular function, coronary blood flow, and wall thickening. Myocardial preconditioning was induced by 10 min coronary artery occlusion. After 24 h of reperfusion (during the SWOP), the hearts were excised. Coronary microvessels were isolated and incubated in presence of 1) the endothelium-dependent agonists carbachol and bradykinin, 2) the calcium ionophore A23187, and 3) the angiotensin-converting enzyme (ACE) inhibitors enalaprilat and ramiprilat. Nitrite, a metabolite of NO, was measured. Under baseline conditions, nitrite production in microvessels from SWOP was 30% higher than that from normal (96 +/- 4 vs. 74 +/- 3 pmol/mg, P < 0.01, respectively). Nitrite production in response to carbachol, bradykinin, and A23187 was also enhanced in microvessels from SWOP (P < 0.05). These enhanced responses were abolished by N(G)-nitro-l-arginine methyl ester (l-NAME) or the endothelial receptor-specific antagonists atropine and HOE-140. The level of eNOS protein in the SWOP myocardium was twofold higher than that in the non-SWOP myocardium. Nitrite production in response to the ACE inhibitors was greater in microvessels from SWOP. These effects were blocked by l-NAME, HOE-140, or dichloroisocoumarin (which inhibits kinin formation). We found that a brief ischemic episode induced delayed, enhanced NO production in coronary microvessels and an upregulation of eNOS protein. These findings suggest that eNOS is a mediator during the SWOP. The ability of ACE inhibitors to enhance NO release during the SWOP points to an additional clinical application for these drugs.     

Osmotic reflection coefficient for total plasma protein in lung microvessels

The osmotic reflection coefficient (sigma) for total plasma proteins was estimated in 11 isolated blood-perfused canine lungs. Sigma's were determined by first measuring the capillary filtration coefficient (Kf,C in ml X min-1 X 100g-1 X cmH2O-1) using increased hydrostatic pressures and time 0 extrapolation of the slope of the weight gain curve. Kf,C averaged 0.19 +/- 0.05 (mean +/- SD) for 14 separate determinations in the 11 lungs. Following a Kf,C determination, the isogravimetric capillary pressure (Pc,i) was determined and averaged 9.9 +/- 0.5 cmH2O for all controls reported in this study. Then the blood colloids in the perfusate were either diluted or concentrated. The lung either gained or lost weight, respectively, and an initial slope of the weight gain curve (delta W/delta t)0 was estimated. The change in plasma protein colloid osmotic pressure (delta IIP) was measured using a membrane osmometer. The measured delta IIP was related to the effective colloid osmotic pressure (delta IIM) by delta IIM = (delta W/delta t)0/Kf,C = sigma delta IIP. Using this relationship, sigma averaged 0.65 +/- 0.06, and the least-squares linear regression equation relating Pc,i and the measured IIP was Pc,i = -3.1 + 0.67 IIP. The mean estimate of sigma (0.65) for total plasma proteins is similar to that reported for dog lung using lymphatic protein flux analyses, although lower than estimates made in skeletal muscle using the present methods (approximately 0.95).     

Evidence for the involvement of a 35-kDa membrane protein in the synthesis of glucosylphosphoryldolichol

Photoactivatable (-²³P)5-azidoUDPGlc binds to two proteins in rat liver microsomes. As determined by SDS-PAGE electrophoresis, the molecular masses of the³²P-labeled proteins were found to be 62-and 35-kDa. Binding of the photoprobe to both proteins was inhibited by addition of unlabeled UDPGlc. Labeling of the higher molecular weight protein occurred in the absence of photoactivation. In contrast, formation of the³²P-labeled 35-kDa protein was dependent on exposure of the membranes to UV light (250nm). Moreover, labeling of the 35-kDa protein required the intact sugar nucleotide and divalent cations and was affected by the level of the endogenous and exogenous dolichylphosphate. All of these results are consistent with the possibility that the 35-kDa membrane protein is a component of glucosylphosphryldolichol synthase.     

A possible mechanism for the increased oxidation of choline after chronic ethanol ingestion

An attempt has been made to determine the location of the site at which the metabolism of ethanol interacts with that of choline to produce an increase in the oxidation of choline. The first enzyme in the oxidation pathway for choline, choline dehydrogenase, was assayed using a newly developed spectro-photometric assay and freshly isolated intact rat liver mitochondria. No changes were observed in either the ‘apparent’ V or the ‘apparent’ K_m values of choline dehydrogenase for choline after ethanol ingestion. However, when the choline oxidase system was assayed, a 28% decrease in ‘apparent’ K_m for choline and a 53% increase in ‘apparent’ V was observed. The effects of ATP on choline oxidase were studied further, and a 29.4% decrease was observed in mitochondrial ATP levels from freshly isolated mitochondria from the ethanoltreated rats. In vitro aging of mitochondria further decreased the level of ATP, and the rate of decrease was considerably faster during the first hour in the mitochondria from the ethanol-treated animals. The decreases in ATP from both control and experimental mitochondria were accompanied by increases in choline oxidase activity. The initial decrease in ATP was correlated with an increase in mitochondrial ATPase activity which may be related to an increase in mitochondrial Mg²⁺. Because chronic ethanol ingestion has resulted in decreased oxidation rates of succinate and β-hydroxybutyrate while at the same time increasing the oxidation rates of choline, the studies reported here suggest that the effect of chronic ethanol ingestion is primarily on a step that is unique to choline and which probably exists prior to the electron transport chain.     

Protein synthesis during right-ventricular hypertrophy after pulmonary-artery stenosis in the dog.

The rate of protein synthesis in the heart of normal dogs and those with pulmonary-artery stenosis was measured by a continuous intravenous infusion of [14C]tyrosine. The protein-synthesis rate of both ventricles was the same in normal dogs and averaged 7.5% per day. The right ventricle hypertrophied rapidly after the acute imposition of pulmonary-artery stenosis, the wet weight increasing by 84% after 24 days, with the rate of increase being most rapid over the first 5 days. The left ventricle remained largely unaffected and served as an internal control. During hypertrophy there was an increased incorporation of [14C]tyrosine into protein without a significant change in the specific radioactivity of free tyrosine in the ventricles. After 5 days of stenosis the synthesis rate of the total mixed proteins of the right ventricle had increased to 13.6% per day, compared with 6.2% in the control left ventricle. This increase in synthesis was reflected in both the myofibrillar and sarcoplasmic proteins. After 5 days the protein-synthesis rate decreased, but still remained significantly elevated above that in the control left ventricle by 24 days of stenosis.     

Left ventricular rupture threshold during the healing phase after myocardial infarction in the dog

The mechanical resistance of the infarcted left ventricle to rupture, or rupture threshold, was measured by the balloon technique 1-42 days after left anterior descending coronary artery ligation in 70 dogs: 26 without infarction (18 sham, 8 with ligation) and 44 with infarction. Rupture threshold in noninfarcted hearts was higher than in infarcted hearts (1168 +/- 165 (SD) vs. 754 +/- 223 mmHg (1 mmHg = 133.32 Pa), p less than 0.001) and did not change over 6 weeks. In contrast, rupture threshold in infarcted hearts decreased (p less than or equal to 0.05) after 14 days, the average value for 21-42 days being less than that for 1-14 days: 577 +/- 140 vs. 867 +/- 191 mmHg, p less than 0.001. Passive left ventricular stiffness in infarcted hearts was higher than for noninfarcted hearts throughout the 6 weeks during early filling (11.1 +/- 3.9 vs. 7.1 +/- 1.4 mmHg/mL, p less than 0.001) but decreased (p less than or equal to 0.05) after 14 days during the prerupture phase (11.3 +/- 5.3 vs. 6.2 +/- 3.0 mmHg/mL, p less than 0.005). Between 7 and 42 days, the infarct zone showed marked increase in hydroxyproline (10.0 +/- 2.0 vs. 48.8 +/- 19.7 mg/g dry weight, p less than 0.001), shrinkage (infarct size, 25 +/- 9 vs. 9 +/- 5% of the left ventricle, p less than 0.005), and thinning (infarct to normal wall thickness ratio, 0.83 +/- 0.11 vs. 0.51 +/- 0.09, p less than 0.001) but little further stretching (expansion index or the ratio of lengths of infarcted and noninfarcted segments, 1.14 +/- 0.10 vs. 1.28 +/- 0.17, p less than 0.2). A mild decrease (p less than 0.05) in left atrial pressure and increase (p less than 0.05) in diastolic area and fractional change in area (two-dimensional echocardiography) were detected at 6 weeks. The late decrease in rupture threshold and prerupture stiffness of the infarcted left ventricle and thinning of the scar suggest a late decrease in mechanical strength and resistance of the infarcted left ventricle to distension.