Angiotensinogen genotype and plasma volume in nulligravid women.

OBJECTIVE: To determine if nonpregnant plasma volume is altered in women who are homozygous for the T 235 coding angiotensinogen allele, which predisposes women to an increased risk of preeclampsia. METHODS: We measured plasma volume by Evans blue dilution and analyzed it as a function of angiotensinogen genotype in 15 nulligravid women during midfollicular phase of 26 menstrual cycles. Eleven women were evaluated during two cycles, and four women were evaluated in one cycle. Fourteen women were white, and one was Asian. No subjects had illnesses or were taking medication. The range of body mass index (BMI [kg/m2]) was 20.2-31.0. Plasma volume (mL) was reported as plasma volume divided by BMI to control for variations in body sizes. Statistical analysis was performed by analysis of variance with post hoc testing using Fisher least significant difference test for multiple comparisons (P < .05 accepted for significance). RESULTS: Angiotensinogen genotype analysis showed five women homozygous for M 235, three women homozygous for T 235, and seven women who were heterozygous (MT 235). T 235 homozygotes had significantly lower plasma volume divided by BMI compared with women who were homozygous for M 235 and women who were heterozygous for MT 235 (mean + standard deviation [SD] [71.2 + 8.8, 86.6 + 5.2, 95.8 + 15.6, respectively, P < .05]). There was a tendency toward higher plasma volume in heterozygote MT 235 compared with homozygote M 235 carriers, but it was not statistically significant. CONCLUSION: We conclude that the homozygous T 235 coding angiotensinogen genotype is associated with reduced plasma volume in nulligravid women during the follicular phase of the menstrual cycle compared with M 235 homozygotes and heterozygotes. This association of the T 235 coding genotype might contribute to fetal growth restriction in preeclampsia.

Amniotic fluid glycine-valine ratio and neonatal morbidity in fetal growth restriction.

OBJECTIVE: To test the hypothesis that an elevated amniotic fluid glycine-valine ratio predicts neonatal morbidity in growth-restricted newborns. METHODS: Amniotic fluid (AF) was collected from 122 third-trimester pregnancies (range 31-39 weeks), 49 of which were complicated by fetal growth restriction. Amino acid analysis was performed by high-pressure liquid chromatography. Glycine-valine ratios were compared between normal and growth-restricted fetuses. Neonatal morbidity within the group of growth-restricted fetuses was characterized by evaluation of neonatal hypoglycemia, arterial cord blood gas analysis, and birth weight percentile. We also examined the correlation of AF glycine-valine ratio to the umbilical artery resistance index. The median interval between AF sampling and delivery was 1 day (range 0-8 days). Analyses were performed by Student t test, chi 2 with Yates correction, or simple correlation when appropriate. P < .05 was considered significant. RESULTS: Growth-restricted fetuses have a significantly elevated AF glycine-valine ratio compared with control subjects (3.31 +/- 1.06 versus 2.61 +/- 0.77, respectively, P < .001). There was no association of the glycine-valine ratio with gestational age for either group. An elevated glycine-valine ratio was not associated with neonatal hypoglycemia within the growth-restricted group (hypoglycemia: [n = 16] 3.19 +/- 1.07; no hypoglycemia: (n = 30) 3.44 +/- 1.09). There were no significant correlations of glycine-valine ratio with arterial cord blood pH (r = -0.10), oxygen pressure (r = 0.04), or base deficit (r = 0.12). There were no significant correlations of glycine-valine ratio and birth weight percentile (r = -.24) or umbilical artery resistance index (r = -.14). CONCLUSION: Amniotic fluid glycine-valine ratio is elevated in growth-restricted fetuses compared with control fetuses. However, the level of glycine-valine elevation is not associated with neonatal morbidity related to hypoglycemia, arterial cord blood gas abnormalities, or birth weight percentile.

Hypertrophy of colonic smooth muscle: structural remodeling, chemical composition, and force output.

The cellular basis of adaptations occurring during the development of megacolon was studied with the lethal spotted mouse model. Age-dependent changes in the length-force characteristics of the colon reach a steady state by 3-4 mo and include an increased relative force development at very short muscle lengths. In megacolon the following occur: 1) structural remodeling expressed as a greater increase in the fraction of maximum force production at short lengths, a shift of optimum length (Lo) to longer lengths, and no change in force per square centimeter; 2) hypertrophy and hyperplasia of both circular and longitudinal muscle; 3) high resting compliance consistent with no disproportionate change in collagen or elastin composition; 4) marked distension so that in situ circumference approximately 1.8 Lo, where active force production is low, and 5) slack length approximately 0.65 Lo, as in normal colon. Biochemical remodeling in megacolon includes disproportionate increases in ATP and phosphocreatine concentration, with 3.5-fold more preformed phosphagen than in normal colon. The myosin concentration is the same in both muscles, but the actin concentration is 1.5-fold greater in megacolon. Most of the cellular changes in megacolon would facilitate high active force output from the muscle at much larger intestinal diameters.

In vivo estrogen regulation of epidermal growth factor receptor in human endometrium.

The effects of estrogen and progesterone on the expression of epidermal growth factor receptor (EGFR) in human endometrium were studied in hypogonadal women under conditions that simulated a normal menstrual cycle. All women received the same regimen of estrogen and progesterone and underwent serial biopsies. In one group of women (group I), a biopsy was obtained before receiving estrogen (CD0) and after 11 days (CD11) of estrogen replacement. A second group of women was biopsied on CD11 and CD21 to assess the combined effects of progesterone and estrogen (group II). Immunohistochemistry was used to test for the presence of EGFR, and a ribonuclease protection assay was used to assess the amounts of EGFR messenger ribonucleic acid (RNA) relative to ribosomal RNA in the tissue. In group I, a significant increase in EGFR messenger RNA from CD0 to CD11 was observed. A similar increase was observed to occur between CD11 and CD21 in group II. Immunostaining for EGFR was absent in all CD0 biopsies, but was present in all estrogen-exposed endometrium. No difference in immunostaining was noted between CD11 and CD21. We conclude that estrogen stimulates the synthesis of EGFR in human endometrium and that progesterone does not appear to modulate this effect. The examination of other parameters in hormone-replaced hypogonadal subjects will be valuable in understanding the complex physiological regulation of the human endometrium.

Oxygen-induced changes in protein synthesis and cell proliferation in cultured lung slices.

Elevated fractions of inspired O2 induce significant remodeling of the airways and vasculature of the lung. The present study was undertaken to determine the direct effects of altered levels of O2 on protein synthesis and cell proliferation in lung tissue cultured in vitro. Rat lungs were inflated with low-melt agarose, cut transversely into 1-mm sections, and cultured in a serum-free medium for up to 7 days in the presence of 10, 21, 40, or 70% O2. Tissue structure integrity was maintained as assessed by light and electron microscopy. Fractional synthesis rates (FSR, %protein/day) of soluble protein from cultured lung homogenates demonstrated an O2 concentration-dependent response. Tissue cultured in the presence of 70% O2 exhibited the highest FSR. The FSR of tissue cultured in 21 or 40% O2 did not differ and demonstrated FSR values greater than tissue cultured in 10% O2. Cell proliferation was assessed histologically in parenchymal gas-exchange regions of lung slices cultured in the presence of 5-bromo-2'-deoxyuridine. Labeling indexes for tissue cultured in 21, 40, or 70% indicated an O2-dependent increase in cell proliferation after 3 days in culture followed by a return to baseline levels after 7 days. Tissue cultured in the presence of 10% O2 showed no change in cell proliferation over time. The data indicate a direct influence of O2 on lung cell growth and proliferation. Additionally, these studies show that this in vitro model may be suitable for further understanding of the mechanistic basis involved in proliferative events during lung injury.

Increased collagen synthesis and decreased collagen degradation in right ventricular hypertrophy induced by pressure overload.

OBJECTIVE: The aim was to examine the effect of pressure overload in rabbits on ventricular collagen metabolism and procollagen gene expression. METHODS: Right ventricular hypertrophy was induced by banding the pulmonary artery such that the diameter of the vessel was reduced by 50%, and animals killed in groups after two and 14 days. Collagen synthesis and degradation of newly synthesised collagen were assessed following a single intravenous injection of 3H-proline with a flooding dose of non-radioactive proline, given 3 h before the animals were killed. Northern and slot blot analyses were performed to measure procollagen alpha 1(I) mRNA. RESULTS: The fractional collagen synthesis rate increased sixfold in the right ventricle only 2 d after pulmonary artery banding (p < 0.001), then fell to just over double the control value by 14 d (p < 0.05 from control). The proportion of newly synthesised collagen degraded decreased from 50.7(SD 12.8)% to 26.8(15.8)% in 2 d (p < 0.05) and remained at this level. The procollagen alpha 1(I) mRNA level increased by more than fourfold in the right ventricle 2 d after the onset of pressure overload, and was less than three times control levels at 14 d. CONCLUSIONS: The development of right ventricular hypertrophy is associated with a rapid increase in collagen production, with regulation at multiple sites in the biosynthetic pathway. This regulation occurs at both transcriptional and post translational levels.

Precursor pools of protein synthesis: a stable isotope study in a swine model.

The accuracy of using other free pools in lieu of tRNA for calculation of tissue protein synthesis in liver (L), skeletal muscle (SM), and heart (H) was assessed in six adult miniature swine using L-[1-13C]leucine and L-[ring-2H5]phenylalanine as tracers. L leucyl-tRNA enrichment was higher than arterial plasma leucine and ketoisocaproate (KIC) enrichments, and L phenylalanyl-tRNA enrichment was higher than arterial phenylalanine enrichment (P < 0.05). No such differences were noted in SM and H. Leucyl- and phenylalanyl-tRNA enrichments in L were best predicted by the respective amino acid enrichments in tissue fluid [TF; Leu: slope (m) = 0.954 +/- 0.035; Phe: m = 1.011 +/- 0.032] using linear regression analysis to determine the accuracy of the prediction, whereas plasma phenylalanine reasonably predicted phenylalanyl-tRNA (artery: m = 0.821 +/- 0.032; vein: m = 0.947 +/- 0.135). In SM, plasma KIC (artery: m = 0.846 +/- 0.046; vein: m = 0.881 +/- 0.043) and TF leucine (m = 0.788 +/- 0.034) predicted leucyl-tRNA with high accuracy. In H tissue, TF (m = 0.991 +/- 0.044) was the best predictor of leucyl-tRNA enrichment, whereas arterial phenylalanine (m = 0.912 +/- 0.015) was the most reliable predictor of phenylalanyl-tRNA enrichment. The relationships between aminoacyl-tRNA and other free pools in the same species under the same study conditions differ in different tissues. Use of KIC in lieu of leucyl-tRNA for calculating muscle protein synthesis is supported by this study.

Isolation of myosin heavy chain from small skeletal muscle samples by preparative continuous elution gel electrophoresis: application to measurement of synthesis rate in human and animal tissue.

A convenient procedure for the isolation of milligram quantities of myosin heavy chain (MHC) from small samples of muscle tissue using preparative gel electrophoresis is described. Application of the methodology to measurements of the fractional synthesis rates (ks) of MHC using skeletal muscle biopsy samples from humans and cardiac tissue from pigs following continuous intravenous infusions of stable isotope (L-[1-13C]-leucine) is demonstrated. The sensitivity and reproducibility of the determination of stable isotope enrichment are also defined. In addition, measurements of the ks of MHC in skeletal muscle of rats in vivo using radioisotope-tracer methodologies are described.

Protein synthesis in pulmonary arteries from rats exposed to hyperoxia.

These studies were undertaken to determine the relationship of early changes in the synthesis rates and contents of collagen, elastin, and soluble tissue protein of pulmonary arteries in rats exposed chronically to normobaric hyperoxia. The growth response of pulmonary arteries was characterized by proportionate increases in the contents of the three protein fractions after 7 days (130% of control) and 21 days (194% of control) of exposure. Fractional rates of protein synthesis were assessed both in vivo and in vitro with the use of several radiolabeled amino acids as tracers to minimize uncertainties of the relationships of the specific radioactivities of measured amino acid pools and the precursors for the proteins fractions. Values for fractional synthesis rates of collagen, elastin, and soluble protein in vitro in pulmonary arteries isolated from control rats were 2.2, 1.6, and 19%/day, respectively. Rates of synthesis of collagen and soluble protein in vitro were approximately 20% lower than that determined in control rats in vivo. The fractional synthesis rates of the three protein fractions in isolated arteries from experimental rats were unchanged after 1 day of hyperoxic exposure, decreased marginally after 3 days, and markedly increased after 7 days. At this time the absolute increments in the fractional synthesis rates of collagen (+4.7%/day) and elastin (+5.0%/day) were less than that of soluble tissue protein (+16%/day) and were more comparable to the accumulation rate of proteins in the tissue. The disproportionate increment in the fractional rate of soluble protein synthesis suggests that the fractional rate of degradation of soluble protein was also increased during the growth response in this model of hypertension.

Composition and mechanics of mesenteric resistance arteries from pregnant rats.

We tested the hypothesis that the systemic resistance vasculature of the rat is remodeled during pregnancy as evidenced by significant alterations in the passive mechanical properties and extracellular matrix proteins in mesenteric arteries. Mechanical characteristics were determined for arteries from 20-day pregnant rats (n = 6) and age-matched controls (n = 5). Lumen diameter and wall thickness were measured in pressurized arteries (250-microns diameter) using a dimension analyzing system. Distensibility (the relative change in diameter per unit change in pressure) was less in the arteries from the pregnant rats (P less than 0.01). The calculated stress-strain relationships and elastic moduli indicated that the arteries were less stiff by late gestation (P less than 0.05). Ultramicro amino acid analysis and radioimmunoassay were used to measure hydroxyproline, desmosine, and leucine as indicators of collagen, elastin, and total protein, respectively, in similar-sized arteries. Hydroxyproline/leucine (index of collagen) and desmosine/leucine (elastin concentration) decreased 19 and 15% by late gestation (P less than 0.05). The significant alterations in passive mechanics and in extracellular protein content support the concept that arterial wall remodeling in the peripheral vasculature may be one component of the cardiovascular adaptations during pregnancy.

Amniotic fluid and plasma glycine/valine ratios in substrate deprived growth retarded fetal rats.

Characteristic profiles of the free amino acid concentration in umbilical cord blood of growth retarded newborns have been observed. We hypothesized that the amniotic fluid of growth retarded fetal rats would show an increase in the ratio between glycine and valine which would parallel the pattern observed in the cord blood of growth retarded neonates, thus providing an index for the antepartum identification of the substrate deprived growth retarded fetus. Six test and 6 control dams were tested. Four fetuses per dam, matched for uterine location were examined. Test animals were fasted for 72 hours. Sampling was performed on day 21 under anaesthesia. Fetal size was significantly reduced (P < 0.0001) in the test group. [T = 2.68 gs. +/- 0.28 vs. C = 3.67 gs. +/- 0.25]. Fetal plasma concentrations of glycine showed an increase in test animals (P < 0.01) while valine showed a significant reduction (P < 0.0001). Glycine (pm/microliters) T = 308 +/- 64 vs. C = 269 +/- 47, valine (pm/microliters) T = 424 +/- 79 vs. C = 671 +/- 218]. Amniotic fluid concentrations for both glycine and valine were significantly decreased (P < 0.0001) in test animals. [Glycine (pm/microliters) T = 710 +/- 124 vs. C = 931 +/- 178; valine (pm/microliters) T = 845 +/- 169 vs. C = 1,339 +/- 234]. The glycine/valine ratio was significantly increased (P < 0.01) in both fetal plasma and amniotic fluid in test animals [Plasma T = 0.74 +/- 0.18 vs. C = 0.43 +/- 0.13. Amniotic fluid T = 0.85 +/- 0.08 vs. C = 0.69 +/- 0.09]. Consistent with our hypothesis, the amniotic fluid concentrations generally parallel the observations made in the plasma. This finding could enhance the antepartum identification of the substrate deprived growth retarded fetus.

In vivo measurement of myocardial protein turnover using an indicator dilution technique.

We applied a nondestructive tracer technique, previously developed for measuring skeletal muscle protein turnover, to the measurement of myocardial protein turnover in vivo. During a continuous infusion of L-[ring-2,6-3H]phenylalanine to anesthetized, overnight-fasted dogs, we measured the uptake of radiolabeled phenylalanine from plasma and the release of unlabeled phenylalanine from myocardial proteolysis using arterial and coronary sinus catheterization and analytic methods previously applied to skeletal muscle. Using these measurements, together with a model of myocardial protein synthesis that assumes rapid equilibration of tracer specific activity between myocardial phenylalanyl-tRNA and circulating phenylalanine, we estimated the rates of heart protein synthesis and degradation. The rate of heart protein synthesis was also estimated directly from the incorporation of labeled phenylalanine into tissue protein. The use of [3H]phenylalanine was compared with L-[1-14C]leucine in the measurement of heart protein turnover in dogs given simultaneous infusion of both tracers. Leucine uptake and release by the myocardium exceeded that of phenylalanine by 3.1 +/- 0.4- and 1.7 +/- 0.3-fold, respectively, consistent with leucine's 2.4-fold greater abundance in heart protein and its metabolism via other pathways. Phenylalanine is the preferred tracer for use with this method because of its limited metabolic fate in muscle. One theoretical limitation to the method, slow equilibration of circulating labeled phenylalanine with myocardial phenylalanyl-tRNA, was resolved by comparison of these specific activities after a 30-minute infusion of labeled phenylalanine in the rat. A second, empirical limitation involves precision in the measurement of the small decrements in phenylalanine specific activity that occur with each pass of blood through the coronary circulation. This was addressed by improving the precision of both the measurements of phenylalanine concentration and phenylalanine specific activity using high-performance liquid chromatography. We conclude that the in vivo measurement of phenylalanine tracer exchange across the myocardium permits the nondestructive estimation of heart protein turnover in the intact animal.

Changes in collagen and elastin in rabbit right-ventricular pressure overload.

Collagen content, the ratio of collagen types I and III and elastin content were measured in 5-6- and 10-12-week-old rabbits with and without right-ventricular pressure overload. Significant and equivalent hypertrophy occurred in both age groups. A 2-day pressure overload caused a fall in collagen concentration below control levels in right-ventricular tissue from the older animals, but no change in the younger ones. A 2-week pressure overload in the older animals resulted in a rise in collagen concentration, a decreased ratio of type III to type I plus III [III/(I + III)] collagens, a fall in desmosine concentration and a fall in the desmosine/hydroxyproline ratio in the right ventricle. None of these changes occurred in the younger age group. We hypothesize that the changes in connective-tissue proteins after overload in the older group may contribute to previously observed changes in mechanical performance. The divergent connective-tissue responses in the two groups suggest the importance of age in determining outcome, as well as the possibility of separate regulatory mechanisms for contractile and for architectural elements of the heart.

Regulation of myosin heavy-chain gene expression during skeletal-muscle hypertrophy.

Changes in the myosin phenotype of differentiated muscle are a prominent feature of the adaptation of the tissue to a variety of physiological stimuli. In the present study the molecular basis of changes in the proportion of myosin isoenzymes in rat skeletal muscle which occur during compensatory hypertrophy caused by the combined removal of synergist muscles and spontaneous running exercise was investigated. The relative amounts of sarcomeric myosin heavy (MHC)- and light (MLC)-chain mRNAs in the plantaris (fast) and soleus (slow) muscles from rats was assessed with cDNA probes specific for different MHC and MLC genes. Changes in the proportion of specific MHC mRNA levels were in the same direction as, and of similar magnitude to, changes in the proportion of myosin isoenzymes encoded for by the mRNAs. No significant changes in the proportion of MLC proteins or mRNA were detected. However, high levels of MLC3 mRNA were measured in both normal and hypertrophied soleus muscles which contained only trace amounts of MLC3 protein. Small amounts of embryonic and neonatal MHC mRNAs were induced in both muscles during hypertrophy. We conclude that the change in the pattern of myosin isoenzymes during skeletal-muscle adaptation to work overload is a consequence of changes in specific MHC mRNA levels.

Efficiency and capacity of protein synthesis are increased in pressure overload cardiac hypertrophy.

We measured the rate of protein synthesis and total RNA content in the right ventricle (RV) at day 2 and day 4 after pulmonary artery constriction to determine the contributions of changes in capacity and efficiency of in vivo protein synthesis to pressure overload (PO) cardiac hypertrophy. A significant increase in the proportion of RV weight to total heart weight was observed at day 2 and day 4 when compared with untreated controls. The rate of protein synthesis was significantly higher at day 2 post-PO (0.31 +/- 0.06 day-1 or 30 +/- 5 mg.g RV-1.day-1, means +/- SD, P less than 0.05) as well as at day 4 (0.25 +/- 0.05 day-1 or 28 +/- 9 mg.g RV-1.day-1, P less than 0.05) than for untreated rabbits (0.15 +/- 0.03 day-1 or 17 +/- 4 mg.g RV-1.day-1). RNA content was significantly higher at day 2 (1.47 +/- 0.17 mg/g RV, P less than 0.05) than in controls (1.16 +/- 0.14 mg/g RV), whereas there was a slight but nonsignificant increase at day 4 (1.36 +/- 0.21 mg/g RV, P less than 0.1). The efficiency of protein synthesis (synthesis/RNA) per gram RV was significantly increased both at day 2 (20.5 +/- 2.2 g protein.g RNA-1.day-1, P less than 0.05) and day 4 (19.8 +/- 3.5 g protein.g RNA-1.day-1, P less than 0.05) compared with control (14.6 +/- 2.3 g protein.g RNA-1.day-1). The increase in efficiency appeared to be caused by pressure overload itself based on a comparison of 0-4 day data vs. data obtained from sham animals (P less than 0.05).

Fractional synthesis rates in vivo of skeletal-muscle myosin isoenzymes.

The synthesis rates of different myosin isoenzymes in a single muscle, and of the same isoenzymes in different muscles (soleus, masseter and plantaris), were measured. The rate of total protein synthesis was significantly higher in the soleus [greater than 95% slow myosin (SM)] than in the plantaris [greater than 95% fast myosin (FM)]. Two fast isoenzymes, FM2 and FM3, were synthesized at different rates in the masseter, and SM was synthesized at a faster rate than FM. Intermediate myosin had a synthesis rate similar to that of FM. There was a small but significant difference between the synthesis rates of the SM isoenzymes of the soleus and masseter muscles. FM3 was synthesized faster in the masseter than in the plantaris, whereas FM2 was synthesized faster in the plantaris than in the masseter.

Myosin isozyme synthesis and mRNA levels in pressure-overloaded rabbit hearts.

The in vivo synthesis rates of myosin isozyme heavy chains beta and alpha were measured in right ventricular (RV) muscle at 2 and 4 days following pulmonary artery constriction in rabbits, together with measurements of their relative mRNA levels. The synthesis rate of beta-myosin heavy chains was elevated in 2-day (0.27 +/- 0.06 day-1 or 2.5 +/- 0.7 mg/g RV/day, mean +/- SD) and in 4-day (0.25 +/- 0.08 day-1 or 2.8 +/- 1.0 mg/g RV/day) pressure overload, when compared to untreated rabbits (0.15 +/- 0.04 day-1 or 1.5 +/- 0.4 mg/g RV/day). However, the synthesis rates of alpha-myosin heavy chains in the same hearts were not altered significantly. There was a differential increase in the fractional synthesis rate of beta vs. alpha heavy chains in 2-day and 4-day pressure overload and in 2-day shams, suggesting switching toward beta heavy chain synthesis had occurred at these time points. beta heavy chain synthesis, as a proportion of total (alpha + beta) heavy chain synthesis, was significantly higher in 4-day pressure overload (78 +/- 9%) than in 4-day sham rabbits (63 +/- 6%). This increase in relative beta-synthesis was associated with a significant increase in the relative proportion of beta heavy chain mRNA level (76 +/- 13% vs. 56 +/- 7%). Furthermore, relative beta-synthesis and the beta-mRNA levels correlated linearly with each other in all experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in skeletal-muscle myosin isoenzymes with hypertrophy and exercise.

The patterns of myosin isoenzymes in fast- and slow-twitch muscles of the rat hindlimb were studied, by pyrophosphate/polyacrylamide-gel electrophoresis, with hypertrophy (induced by synergist removal) and with spontaneous running exercise of 4 and 11 weeks duration. At 11 weeks, changes with hypertrophy in the slow-twitch soleus, composed of greater than 95% SM2 (slow myosin 2) in normal muscles, were minor, and consisted of an increase in the SM1 and SM1', and a loss of intermediate myosin (IM), an isoenzyme characteristic of Type IIa fibres [Fitzsimons & Hoh (1983) J. Physiol. (London) 343, 539-550]. The changes were dramatic, however, in the fast-twitch plantaris muscle. There was a 3-fold increase in the proportion of SM. In addition, IM became the predominant isoenzyme in the profile of hypertrophied plantaris by 4 weeks. These increases were balanced by decreases in the proportion of FM2 (fast myosin 2), with FM1 completely absent from the profile at 11 weeks. The changes in the plantaris with exercise were similar in direction but not as extensive as those with hypertrophy, and FM1 remained present at control levels throughout the study. When hypertrophy and exercise were combined, the increase in slow myosin was equal to the sum of the increases with each treatment alone. Changes at 4 weeks were intermediate between those of control and 11-week muscles. Peptide mapping of individual myosin isoenzymes showed that the heavy chains of IM were different from either fast or slow heavy chains. Furthermore, IM was found to be composed of a mixture of fast and slow light chains. These changes suggest that a transformation of myosin from fast to slow isoforms was in progress in the plantaris in response to hypertrophy, via a Type-IIa-myosin (IM) intermediate stage, a phenomenon similar to that occurring in chronically stimulated fast muscles during fast-to-slow transformation [Brown, Salmons & Whalen (1983) J. Biol. Chem. 258, 14686-14692].

Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts.

Compartmentation of the amino acid precursor pools for protein synthesis in cultured cells can substantially complicate measurements of synthesis rates. This is particularly true for nonessential amino acids such as proline, an amino acid often used in isotopic form to measure collagen synthesis. We have made a comparative study of this problem in cultured IMR-90 fibroblasts using isotopic proline and leucine to measure total protein and collagen synthesis. 3H-leucine in the extracellular (EC) medium equilibrates with tRNA-leucine at an EC concentration of 0.4 mM in both dividing and stationary cells. Thus, under these experimental conditions there is no complicating compartmentation of leucine for protein synthesis. Equilibration of EC and tRNA-bound 3H-proline, however, does not occur even when the EC concentration is in the mM range, based upon simultaneous measurements of synthesis rates using 3H-proline and 3H-leucine together. Furthermore, significant changes in EC proline concentration and specific activity occur over short time intervals (2 hr) if the initial EC proline concentration is below 0.2 mM. Thus, the use of isotopic proline to measure protein synthesis introduces substantial interpretive problems. Serum deprivation causes changes in both total collagen synthesis and the percent of protein synthesis devoted to collagen when measured with either 14C-leucine or 3H-proline. At the same time, isotopic proline remains the better choice for measuring percent collagen synthesis.