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Molecular cloning and ligand binding studies have shown the alpha 2 class of adrenergic receptor (alpha 2-AR) to be a family of at least three related subtypes in humans. These studies have not, however, identified distinct subtype-specific functions for these receptors in vivo. It should be possible to extend the analysis of alpha 2-AR subtype function to the animal level through the use of experimental mammalian embryology in mice. To begin this process, we have isolated two mouse genomic clones encoding alpha 2-AR subtypes and expressed these genes in COS-7 cells for binding studies. Sequence homology and ligand binding data allow the assignment of one clone (M alpha 2-4H) as the mouse homolog of the human alpha 2-C4 subtype. The other clone (M alpha 2-10H) closely resembles the human alpha 2-C10 subtype in sequence but binds with significantly lower affinity to yohimbine and rauwolscine, members of a distinct class of bulky alpha 2-selective antagonists commonly used to evaluate alpha 2-AR function in vivo. To define the domain(s) responsible for this unusual binding property, we constructed a series of M alpha 2-10H/human alpha 2-C10 chimeric receptors. Analysis of these receptors identified a conservative Cys201 to Ser201 change in the fifth transmembrane domain of M alpha 2-10H as being responsible for the low affinity of the mouse receptor for yohimbine.

Protein synthesis and degradation and net uptake and release of amino acids and minerals were investigated in the perfused hemicorpus of acutely uremic and control Sprague-Dawley rats. Rats underwent bilateral nephrectomy or sham surgery and were studied 30 hr after surgery. The uremic rats displayed greater urea N appearance (net urea generation), lower plasma and muscle concentrations of most amino acids, and increased muscle protein degradation as compared to control rats. Muscle protein synthesis was slightly but not significantly decreased in the uremic animals. There was greater net release of phenylalanine, tyrosine, alanine, total nonessential amino acids, total amino acids, potassium and phosphorus from the perfused hemicorpus of uremic rats and greater release of citrulline from sham rats. Muscle ATP, creatine phosphate, cyclic-AMP, and activities of cathepsin B1, cathepsin D, and alkaline protease were not different in the uremic and sham rats. These data provide evidence that acutely uremic rats sustain increased muscle protein wasting which is due to enhanced protein degradation. The increased protein degradation does not appear to be due to enhanced activities of muscle cathepsin B1, cathepsin D or alkaline protease.

The age at which phenylalanine (PA) restricted diet can be safely terminated in phenylketonuria (PKU) children has been the subject of discussion but a recent United States collaborative study has provided data showing that blood PA level should be kept below 10 mg/dl at least through adolescence. This presents nutritional problems in providing an adequate diet to meet the adolescent growth spurt in the absence of reliable information on the nutrient requirements of PKU adolescents. The problem is complicated by the emotional development of adolescence presenting compliance difficulties. The use of a computer programme to calculate nutrient intake and appropriate amino acid restricted diets for adolescents is described. It has been shown using the computer programme that an amino acid mixture specially adapted for use by adolescents is needed to provide sufficient vitamins, minerals and trace elements. Nutritional deficiency in adolescent PKU children may be a factor contributing to developmental abnormality.

Protein synthesis and degradation and net uptake and release of amino acids and minerals were examined in the perfused hemicorpus of bilaterally nephrectomized and sham-operated control rats. Animals were studied 30 h after surgery. In comparison with controls, uremic rats had greater urea N appearance (net urea generation) and lower plasma and muscle concentrations of most amino acids. Muscle protein synthesis was not altered, but protein degradation was greater in uremic versus sham rats. There was greater net release of phenylalanine, tyrosine, alanine, total nonessential amino acids, total amino acids, potassium, and phosphorus from the perfused hemicorpus of uremic rats and greater release of citrulline from sham rats. ATP, creatine phosphate, cAMP, and activities of cathepsin B1, cathepsin D, and alkaline protease were not different in muscles of the uremic versus sham rats. Thus, in acutely uremic rats there is increased protein wasting in the hemicorpus due to enhanced protein degradation. The enhanced protein degradation does not appear to be due to increased muscle cathepsin B1, cathepsin D, or alkaline protease activities.

Protein synthesis and degradation and net uptake and release of amino acids and minerals were investigated in the perfused hemicorpus of acutely uremic and sham-operated control Sprague-Dawley rats. Rats underwent bilateral nephrectomy or sham surgery and were studied 30 hours after surgery. The uremic rats displayed greater urea nitrogen appearance (net urea generation), lower plasma and muscle intracellular concentrations of most amino acids, and increased protein degradation in the hemicorpus as compared with control animals. Muscle protein synthesis was slightly but not significantly decreased in the uremic animals as compared with controls. There was greater net release of phenylalanine, tyrosine, alanine, total nonessential amino acids, total amino acids, potassium, and phosphorus from the perfused hemicorpus of uremic rats and greater release of citrulline from sham rats. Muscle ATP, creatine phosphate, and cyclic AMP, and muscle cathepsin B1, cathepsin D, and alkaline protease activities were not different in the uremic and control rats. These data provide evidence that acutely uremic rats have increased muscle protein wasting which is due to enhanced protein degradation. The cause of the increased muscle protein degradation is unknown.

Red cell and plasma amino acid concentrations were measured in nine healthy subjects, nine patients with advanced chronic renal failure, and before and after dialysis in eight patients undergoing maintenance hemodialysis. Blood was obtained after an overnight fast except for postdialysis specimens. In red cells from the chronically uremic patients and in predialysis specimens, there was increased histidine, cystine, glutamic acid, glycine, ornithine, citrulline, taurine, and N tau-methylhistidine, and an increased glycine to serine ratio. Red cell valine, tyrosine, and the ratios of tyrosine to phenylalanine and valine to glycine were decreased in the two groups. Many amino acid levels that were abnormal in red cells were also abnormal in plasma. However, several amino acids were altered in only one of these compartments. Some red cell or plasma amino acid concentrations were abnormal in the uremic or hemodialysis patients but not in both. During hemodialysis, red cell amino acids did not decrease as markedly as did the plasma concentrations. These findings indicate that in chronically uremic and hemodialysis patients there is an abnormal amino acid pattern in both red cells and plasma. Although the altered amino acid patterns in these two compartments have similarities, they are not identical.

Chronic renal failure is associated with many abnormalities in plasma amino acids. Since patients with renal failure are frequently deficient in vitamin B-6, this study examined whether vitamin B-6 deficiency may be a cause of any of the abnormal plasma amino acid concentrations observed in chronic renal failure. Sham-operated and chronically azotemic rats were pair-fed diets deficient in or replete with vitamin B-6 for 21 d. By the end of 21 d, the EGOT index rose significantly in the B-6-deficient rats but not in the B-6-replete animals. There were several differences in plasma amino acid concentrations between azotemic and control rats. Azotemia and B-6 deficiency each lowered the plasma serine concentration and raised the glycine-to-serine ratio. Plasma glycine was affected by a two-way interaction between azotemia and vitamin B-6 deficiency whereby the highest values were found in the sham-operated vitamin B-6-deficient animals. Plasma alanine and asparagine were reduced by B-6 deficiency and unchanged by azotemia. These results suggest that vitamin B-6 deficiency may contribute to several of the abnormalities in the plasma aminograms observed in chronic renal failure.