The phosphorylation of adrenal proteins in response to adrenocorticotropic hormone

The phosphorylation of rat adrenal protein components in response to adrenocorticotropin has been studied in adrenal quarters, isolated cells, and in vivo. In adrenal quarters, adrenocorticotropic hormone (ACTH)-stimulated phosphorylation or dephosphorylation of proteins was not affected by the presence of protein synthesis inhibitors despite a total inhibition of steroidogenesis. (The term dephosphorylation refers to an apparent decrease in the labeling of a particular protein with 32P at various times after the addition of ACTH. This may be due to enzymatic removal of phosphate or protein degradation or complexation of this protein with another cellular component.) Studies with isolated cell preparations identified several proteins that are phosphorylated or dephosphorylated in response to hormone. These changes in phosphorylation were also observed in adrenal quarters and correlated well with ACTH-stimulated steroidogenesis as determined by temporal analysis and dose-response studies of corticosterone production. In vivo injection of male hypophysectomized rats with [32P]phosphate and ACTH demonstrated changes in the labeling of six adrenal proteins. Many of the proteins phosphorylated in vivo were also demonstrated to be phosphorylated in both in vitro systems. Finally, the injection of a physiological dose of ACTH appeared to selectively activate the type I cAMP-dependent protein kinase within the microsomal fraction as determined by the binding of a photoaffinity-labeled reagent. These results suggest that alterations in phosphorylation of adrenal proteins in response to ACTH is proximal to or independent of the obligatory role of protein synthesis in acute steroidogenesis.     

Optimizing purebred selection for crossbred performance using QTL with different degrees of dominance

A method was developed to optimize simultaneous selection for a quantitative trait with a known QTL within a male and a female line to maximize crossbred performance from a two-way cross. Strategies to maximize cumulative discounted response in crossbred performance over ten generations were derived by optimizing weights in an index of a QTL and phenotype. Strategies were compared to selection on purebred phenotype. Extra responses were limited for QTL with additive and partial dominance effects, but substantial for QTL with over-dominance, for which optimal QTL selection resulted in differential selection in male and female lines to increase the frequency of heterozygotes and polygenic responses. For over-dominant QTL, maximization of crossbred performance one generation at a time resulted in similar responses as optimization across all generations and simultaneous optimal selection in a male and female line resulted in greater response than optimal selection within a single line without crossbreeding. Results show that strategic use of information on over-dominant QTL can enhance crossbred performance without crossbred testing.     

Skeletal muscle sarcolemma in malignant hyperthermia: evidence for a defect in calcium regulation

Sarcolemmal properties implicated in the skeletal muscle disorder, malignant hyperthermia (MH), were examined using sarcolemma-membrane vesicles isolated from normal and MH-susceptible (MHS) porcine skeletal muscle. MHS and normal sarcolemma did not differ in the distribution of the major proteins, cholesterol or phospholipid content, vesicle size and sidedness, (Na+ + K+)-ATPase activity, ouabain binding, or adenylate cyclase activity (total and isoproterenol sensitivity). The regulation of the initial rates of MHS and normal sarcolemmal ATP-dependent calcium transport (calcium uptake after 1 min) by Ca2+ (K1/2 = 0.64-0.81 microM), calmodulin, and cAMP-dependent protein kinase were similar. However, when sarcolemmal calcium content was measured at either 2 or 20 min after the initiation of active calcium transport, a significant difference between MHS and normal sarcolemmal calcium uptake became apparent, with MHS sarcolemma accumulating approximately 25% less calcium than normal sarcolemma. Calcium transport by MHS and normal sarcolemma, at 2 or 20 min, had a similar calmodulin dependence (C1/2 = 150 nM), and was stimulated to a similar extent by cAMP-dependent protein kinase or calmodulin. Halothane inhibited MHS and normal sarcolemmal active calcium uptake in a similar fashion (half-maximal inhibition at 10 mM halothane), while dantrolene (30 microM) and nitrendipine (1 microM) had little effect on either MHS or normal sarcolemmal calcium transport. After 20 min of ATP-supported calcium uptake, 2 mM EGTA plus 10 microM sodium orthovanadate were added to initiate sarcolemmal calcium efflux. Following an initial rapid phase of calcium release, an extended slow phase of calcium efflux (k = 0.012 min-1) was similar for both MHS and normal sarcolemma vesicles. We conclude that although a number of sarcolemmal properties, including passive calcium permeability, are normal in MH, a small but significant defect in MHS sarcolemmal ATP-dependent calcium transport may contribute to the abnormal calcium homeostasis and altered contractile properties of MHS skeletal muscle.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Influence of maternal mineral deficiency on the hepatic metallothionein and zinc in newborn rats

The effects of maternal Zn, Cu, or Fe deficiencies during late gestation on hepatic levels of metals and metallothionein (MT) and the binding of Zn and Cu to protein fractions were investigated in newborn rats. Timed pregnant rats were fed one of the following diets: Zn deficient (Zn-D), Cu-D, Fe-D, or control from day 12 of gestation until birth. The specific nutritional deficiency status of the dams was confirmed by low plasma levels of the deficient metal. Livers from pups were analyzed for MT, metal content, and metal-protein binding. Maternal Zn-D resulted in a greater than 50% reduction of hepatic MT levels in pups, whereas Cu-D and Fe-D had no significant effects. Pups in each deficient group showed a significant decrease in the hepatic levels of the respective metals. Fractionation of hepatic cytosols from the pups by Sephadex G-75 gel filtration showed that in both Fe-D and Cu-D pups the respective metals were depleted from the high molecular weight protein fraction, whereas in Zn-D pups the Zn was depleted mainly from the MT fraction (Ve/V0 approximately 2). Incorporation of [35S] cysteine into MT fractions was significantly lower in Zn-D pups as compared with control pups. These results indicate that there is a specific effect of the maternal Zn-D on the hepatic storage of Zn as MT in newborn rats.     

Calcium-Dependent 4-aminopyridine stimulation of protein phosphorylation in squid optic lobe synaptosomes

When intact synaptosomes were incubated with [gamma-32P]ATP, maximal protein phosphorylation was attained 2 min after the start of incubation. Protein phosphorylation under basal conditions was dependent on external Ca2+, and the dominant peak of phosphorylation was a 50-kd protein. Incubation of intact synaptosomes in the presence of 3-6 mM 4-aminopyridine (4-AP) caused a markedly enhanced phosphorylation of high molecular weight proteins of 90, 100, 130, and 180 kd, with no increase in the 50 or 38 kd proteins. This effect of 4-AP was dependent on external calcium ions in the incubation medium. The 4-AP effect on the high molecular weight proteins was also found in synaptosomal plasma membranes isolated from the synaptosomes. Tetraethylammonium (TEA) ions did not produce this enhancement of phosphorylation.