Regional Rates Of Protein Synthesis Research Articles

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo.

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Double-tracer autoradiographic study of protein synthesis and glucose consumption in rats with focal cerebral ischemia

A double-tracer autoradiographic method for simultaneous measurement of regional glucose utilization (rCMRglc) and regional protein synthesis (PS) in consecutive brain sections is described and applied to study the metabolism of the ischemic penumbra 2 h after occlusion of the middle cerebral artery (MCAO) in rats. In halothane anesthesia, the left middle cerebral artery was permanently occluded. Two hours after MCAO an i.v. bolus injection of 14C-deoxyglucose and 3H-leucine was given and circulated for 45 min. Two sets of brain sections were processed for quantitative autoradiography. Neighboring brain sections exposed an X-ray film (3 H-insensitive), and a 3H-sensitive for determination of rCMRglc and PS, respectively. Sections for PS determination were washed in trichloroacetic acid (TCA) prior to film exposure in order to remove 14C-deoxyglucose and unincorporated 3H-leucine. Regional rates of PS and glucose utilization were measured by densitometric image analysis. Normal rates of metabolism were defined as mean ± 2 SD of values in the non-ischemic cortex. The volumes of ischemic cortex displaying normal rates of PS and glucose utilization, respectively, were measured. The cortical volume with normal PS was significantly less than that of normal rCMRglc: 142 (127-147) mm3 vs. 203 (184-206) mm3. Treatment with the glutamate antagonists MK-801 (1 mgkg–1) and NBQX (30 mg kg–1 x 2) did not significantly change this, although MK-801 tended to reduce the size of the metabolic penumbra calculated as the difference between ischemic cortex with reduced PS and ischemic cortex with reduced rCMReic. [Neurol Res 1991; 21: 687–694]

Effects of aging on regional rates of cerebral protein synthesis in the Sprague-Dawley rat: examination of the influence of recycling of amino acids derived from protein degradation into the precursor pool

The quantitative autoradiographic l-[1- 14C]leucine method for determination of regional rates of cerebral protein synthesis (1CPS leu) requires knowledge of the degree of recycling of leucine derived from protein degradation into the precursor pool for protein synthesis. The influence of recycling can be evaluated by measuring λ, the steady state ratio of the leucine specific activity in the precursor amino acid pool (tRNA-bound leucine) to that in the arterial plasma. To define the changes in 1CPS leu during the process of normal aging in the rat we have evaluated λ in middle-aged (14 months) and aged (24 months) rats and compared its values with those obtained previously in young adult rats (two months of age). The results show that the value of λ is the same in all three age groups, and that there is no change with aging in the fraction of leucine in the precursor pool derived from protein degradation. Our previously reported regional rates of protein synthesis in young adult and aged rats were based on the assumption that there was no recycling of leucine derived from protein degradation into the precursor pool for protein synthesis [Ingvar M. C., Maeder P., Sokoloff L. and Smith C. B. (1985) Brain 108, 155–170]. These values have been recalculated in the present study in order to take into account the appropriate correction for recycling. The recalculated rates are higher than those reported previously, but the effects of aging in the brain as a whole and in some specific brain regions are confirmed. Decreased 1CPS leu was observed by middle-age, and in this cross-sectional study did not appear to decrease further. Of the 39 brain regions examined decreases were found throughout the brain with some proclivity for the brain stem. In comparison with young adults the weighted average rate of protein synthesis in the brain as a whole was found to be decreased by 16 and 11 % in the middle-aged and aged groups, respectively.

Regional cerebral L-[14C-methyl]methionine incorporation into proteins: evidence for methionine recycling in the rat brain.

The specific activity (SA) of free methionine was measured in plasma and in different regions of the rat brain at 15, 30, or 60 min after intravenous infusion of L-[14C-methyl]methionine. Within these time periods, an apparent steady state of labeled free methionine in plasma and in brain was reached. However, the brain-to-plasma free methionine SA ratio was found to be approximately 0.5, showing that an isotopic equilibrium between brain and plasma was not attained. This suggests the presence of an endogenous source of brain free methionine (likely originating from protein breakdown), in addition to the plasma source. The contribution of this endogenous source to the content of free methionine varies significantly among the different brain regions. Our results indicate that the regional rates of protein synthesis measured with L-[11C-methyl]methionine using positron emission tomography would be underestimated, since the local fraction of brain methionine derived from protein degradation would not be considered.

Changes in in vivo rates of protein synthesis on free and membrane-bound polysomes in rat brain during the development of physical dependence on ethanol and after the withdrawal of ethanol

Administration of ethanol and nutrients to rats thrice daily by gavage for 3 days produced a linear increase in physical dependence during the first 3 days and a 2% increase in body weight. Rates of protein synthesis on free and membrane-bound polysomes in whole brain and in 7 brain regions, comprising the entire brain, were measured in vivo under pool expansion conditions with [ 3H]leucine at intervals during the development and decay of ethanol dependence. During dependence development there was a progressive decrease in the rate of protein synthesis on free polysomes, but this change was not significant (P < 0.05) until the third day, and a decrease in the rate on membrane-bound polysomes after 3 days. The inhibition of protein synthesis is attributable to a decreased rate of polypeptide elongation. There was no change in brain weight, DNA content, ribosome content, ribosome distribution on mRNA size. There was, however, a decrease in leucine uptake after 3 days. In an attempt to distinguish between the acute effects of ethanol on regional rates of protein synthesis and those changes associated with dependence development, rates were measured 1.5 h after administering a 5 g/kg dose of ethanol to both control and dependent rats. Rates on free polysomes in the hippocampus-amygdala and thalamus-hypothalamus and on membrane-bound polysomes in the cerebellum and hipocampus-amygdala of dependent rats were reduced; however, there was a general reduction in the rates in control rats that may have obscured reductions in other regions from dependent rats. During early withdrawal, 12 h after the last dose of ethanol, there was an increase in the rate on free polysomes in the pons-medulla and striatum-septum and on membrane-bound polysomes in the hippocampus-amygdala, and a decrease in the rate on free polysomes in the cortex and thalamus-hypothalamus and on membrane-bound polysomes in the cortex. After 24 h, there was an increase in the rate on free polysomes in all regions (cerebellum, cortex, mesencephalon, striatum-septum and thalamus-hypothalamus) except the hippocampus-amygdala and pons-medulla and an increase in the rate on membrane-bound polysomes in all regions (cortex, hippocampus-amygdala, mesencephalon, pons-medulla and striatum-septum) except the cerebellum and thalamus-hypothalamus. The possible relationship of these changes to the homeostat hypothesis of ethanol dependence is discussed.