Measuring Changes In Protein Synthesis In Vivo

Changes in cardiac protein levels occur as a result of perturbations in protein synthesis and/ or degradation. Rates of protein synthesis can be determined using labeled amino acids via measurement of the rate of their incorporation into cardiac proteins. This necessitates characterization of time-course changes in the specific radioactivities of the precursor, i.e., the amino acyl tRNA (StRNA), and the product, i.e., the labeled amino acids in the tissue protein [24], Whilst measuring the specific radioactivity of the protein-bound amino acid presents few practical problems, determining the specific radioactivity of StRNA is more difficult to quantify on a routine basis. This is because tRNA is extremely labile and occurs in low abundance, such that techniques have been devised to approximate StRNA or determine its value indirectly. Frequently, StRNA is represented by either:

(i) the specific radioactivities of the free amino acid in the intracellular pool (measured as the specific radioactivity of the free amino acid in the acid supernatants ofheart homogenates, i.e., Sj), or;

(ii) the specific radioactivity of the free amino acid in the extracellular pool (measured as the specific radioactivity of the free amino acid in the acid supernatant of the plasma, i.e.,Sp).

In animal studies, there are three methods for measuring protein synthesis in vivo, two of which are reliable and in current use. In the pulse tracer injection method, the isotope is injected in small quantities. Figure 17.1 shows that immediately after the pulse injection of the tracer isotope in small laboratory animals, there are complex changes in both Sj and Sp, which makes it difficult to define these curves especially in the initial phase of the radio-labeling period (Phase a). More subtle changes occur in the aftermath of the initial injection period (Phase b) compared to the more dynamic Phase a. However, accurate assessment of protein synthesis necessitates quantifying the area under the entire curve for either Sj or Sp during the radiolabeling period. This involves sacrifice of a large number of animals to characterize the curves for Sj and Sp. As a consequence of the practical constraints inherent in the method, the pulse injection technique is considered unreliable. The 'constant infusion' method facilitates the measurement of protein synthesis rates in single rats. In this method, a tracer amount of amino acid is infused over a few hours (usually between 3-6 hours) in rats. The specific radioactivities rise to a plateau in Phase a, whereas the specific radioactivities ofboth Sj and Sp attain steady state values in the latter period ofPhase b. The disadvantage of this method is that there are large differences between Sj and Sp for tissues with high turnover rates (such as liver and intestine). Furthermore, animals have to be immobilized for up to 3-6 hours, which precludes the use of this method in acute studies. Because of the complex time-course changes in protein turnover and its possible influence on Sj and Sp, the derived value of the fractional synthesis rate (ks) may be in error. This is because, calculation ofks depends on the final Sj and Sp values and the cumulative incorporation of label into proteins, occurring over the 3-6 hour infusion period. For example, we have shown that the rates of muscle protein synthesis in rats measured in the morning (09:00-12:00 hours) are higher than those in the afternoon (15:00-18:00 hours) [25]. This is not easily discernible with the constant infusion method [25],

In thQflooding dose technique, now considered the 'gold standard' method for measuring protein synthesis, the radio-label is injected with a large amount of 'cold' amino acid. With this method there are small decreases in both Sj and Sp, which can in practice be considered to be linear over short labeling periods, such aslO minutes. In the heart, the changes in Sj are quite small (approx. 5%) over 10 mins, and rats are killed at one time-point only. Thus, the features ofboth Phase a and Phase b are identical. Th Qflooding dose method overcomes many of the practical limitations inherent in the constant infusion method. For example, in tissues with high protein turnover rates, the differences between Sj and Sp are minimized with th Qflooding dose method in contrast to the large differences between Sj and Sp noted above. Th Qflooding dose method has been employed in over 600 studies to date, and investigation of heart muscle protein changes include the effects of running, growth hormone, alcohol, intrauterine growth retardation and IGF-I dosage [26-31], In th Qflooding dose method, animals are injected intravenously with phenylalanine (0.150 mmol per lOOg body weight) and sacrificed after 10 minutes [32], Phenylalanine is chosen as this amino acid is (i) not considered to be a regulator of protein synthesis; (ii) it is very soluble; (iii) it occurs in very low concentrations in the intracellular pool, thereby facilitating the flooding phenomena and rapid equilibration between subcellular compartments; (iv) its specific radioactivity can be determined by test-tube techniques. Some studies have injected the labeled isotope intraperitoneally, but this is not recommended as ethanol affects the partitioning of phenylalanine between extracellular and intracellular compartments of free amino acid pools [33], After intravenous injection and allowing a suitable radio-labeling period (usually 10 minutes) fractional rates of cardiac protein synthesis (i.e., percentage of the cardiac protein pool renewed each day, i.e., ks, % per day)

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Figure 17.1 Changes in precursor specific radioactivities in different methods for measuring protein synthesis in vivo. The three methods of measuring protein synthesis are (top) pulse injection of a tracer amount of amino acid, (middle) constant infusion of a tracer amino acids and (bottom) injection of a flooding dose of amino acid. Phase (a) and (b) are arbitrary assigned periods of each method. For a fuller explanation see text. Adapted from works ofP.J. Garlick.

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Figure 17.1 Changes in precursor specific radioactivities in different methods for measuring protein synthesis in vivo. The three methods of measuring protein synthesis are (top) pulse injection of a tracer amount of amino acid, (middle) constant infusion of a tracer amino acids and (bottom) injection of a flooding dose of amino acid. Phase (a) and (b) are arbitrary assigned periods of each method. For a fuller explanation see text. Adapted from works ofP.J. Garlick.

are calculated from the formula: ks= (S^ x 100) /(Sj x t) [32], Where, S^ is the specific radioactivity of the labeled phenylalanine in cardiac protein; Sj is the specific radioactivity of the free amino acid in the intracellular pool; t is the labeling period, in units of a day, i.e., for a 10-min labeling period, 0.00694 days

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