Western blot analysis of BK channel ß1-subunit expression should be interpreted cautiously when using commercially available antibodies.

Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory ß-subunits. There are four ß-subunit subtypes (ß1-ß4), BK ß1-subunit is specific for smooth muscle cells (SMC). Reduced BK ß1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK ß1-subunit reduces channel activity and increases SMC contractility. Several anti-BK ß1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK ß1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK ß1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK ß1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK ß1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK ß1-subunit. The absence of BK ß1-subunit mRNA expression in arteries, colons, and kidneys from BK ß1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK ß1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses.

Effect of intravenous amino acids on glutamine and protein kinetics in low-birth-weight preterm infants during the immediate neonatal period.

Glutamine may be a conditionally essential amino acid in low-birth-weight (LBW) preterm neonates. Exogenously administered amino acids, by providing anaplerotic carbon into the tricarboxylic acid cycle, could result in greater cataplerotic efflux and glutamine de novo synthesis. The effect of dose and duration of amino acid infusion on glutamine and nitrogen (N) kinetics was examined in LBW infants in the period immediately after birth. Preterm neonates (<32 weeks gestation, birth weights 809-1,755 g) were randomized to initially receive either 480 or 960 micromol x kg(-1) x h(-1) of an intravenous amino acid solution for 19-24 hours, followed by a higher or lower amino acid load for either 5 h or 24 h. Glutamine de novo synthesis, leucine N, phenylalanine, and urea kinetics were determined using stable isotopic tracers. An increase in amino acid infusion from 480 to 960 micromol x kg(-1) x h(-1) for 5 h resulted in decreased glutamine de novo synthesis in every neonate (384.4 +/- 38.0 to 368.9 +/- 38.2 micromol x kg(-1) x h(-1), P < 0.01) and a lower whole body rate of proteolysis (P < 0.001) and urea synthesis (P < 0.001). However, when the increased amino acid infusion was extended for 24 h, glutamine de novo synthesis increased (369.7 +/- 92.6 to 483.4 +/- 97.5 micromol x kg(-1) x h(-1), P < 0.001), whole body rate of proteolysis did not change, and urea production increased. Decreasing the amino acid load resulted in a decrease in glutamine rate of appearance (R(a)) and leucine N R(a), but had no effect on phenylalanine R(a). Acutely stressed LBW infants responded to an increase in amino acid load by transiently suppressing whole body rate of glutamine synthesis, proteolysis, and oxidation of protein. The mechanisms of this transient effect on whole body protein/nitrogen metabolism remain unknown.

Amino acids, glutamine, and protein metabolism in very low birth weight infants.

Glutamine has been proposed to be conditionally essential for premature infants, and the currently used parenteral nutrient mixtures do not contain glutamine. De novo glutamine synthesis (DGln) is linked to inflow of carbon into and out of the tricarboxylic acid (TCA) cycle. We hypothesized that a higher supply of parenteral amino acids by increasing the influx of amino acid carbon into the TCA cycle will enhance the rate of DGln. Very low birth weight infants were randomized to receive parenteral amino acids either 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 5 h (AA1.5) or 3.0 g/kg/d for 20 h followed by 1.5 g/kg/d for 5 h (AA3.0). A third group of babies received amino acids 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 20 h (AA-Ext). Glutamine and protein/nitrogen kinetics were examined using [5-(15)N]glutamine, [2H5]phenylalanine, [1-(13)C,15N]leucine, and [15N2]urea tracers. An acute increase in parenteral amino acid infusion for 5 h (AA1.5) resulted in decrease in rate of appearance (Ra) of phenylalanine and urea, but had no effect on glutamine Ra. Infusion of amino acids at 3.0 g/kg/d for 20 h resulted in increase in DGln, leucine transamination, and urea synthesis, but had no effect on Ra phenylalanine (AA-Ext). These data show an acute increase in parenteral amino acid-suppressed proteolysis, however, such an effect was not seen when amino acids were infused for 20 h and resulted in an increase in glutamine synthesis.