inosinic-acid and Acidosis

To understand the mechanisms that initiate the increase in ammonia formation during acute acidosis in kidney [amino-15N]- and [amino-15N]glutamine were used as substrates in isolated perfused rat kidney experiments. Perfused kidneys from methionine sulfoximine-treated rats take up glutamine nitrogen at the rate of 1.50 +/- 0.08 mumol.g kidney-1.min-1 while forming ammonia at a rate of 0.65 +/- 0.09 mumol.g.kidney-1.min-1. Mass spectrometer analysis of the perfusate and urine reveals that ammonia is formed from the amide nitrogen of glutamine at the rate of 0.32 +/- 0.06 mumol.g kidney-1.min-1 and ammonia is formed from glutamate derived from glutamine at the rate of 0.21 +/- 0.04 mumol.g kidney-1.min-1. The balance of the ammonia formed is from unidentified endogenous sources. Addition of HCl to the perfusate to lower perfusate pH increases ammonia formation to 1.09 +/- 0.10 mumol.g kidney-1.min-1. The results exclude a role for the purine nucleotide cycle during acute acidosis and confirm that ammonia formation from glutamate derived from glutamine is via glutamate dehydrogenase. Lowering perfusate pH increases the rate of glutamine deamidation significantly by 0.33 +/- 0.06 mumol.g kidney-1.min-1 and increases the rate of ammonia formation via glutamate dehydrogenase insignificantly by only 0.08 +/- 0.04 mumol.g kidney-1.min-1, whereas ammonia formation from endogenous sources remains unchanged. The results demonstrate that regulation of glutamine deamidation is an important controlling step in ammonia formation during acute metabolic acidosis in kidney.

Topics: Acidosis; Acute Disease; Adenine Nucleotides; Amino Acids; Ammonia; Animals; Gas Chromatography-Mass Spectrometry; Glutamine; Guanine Nucleotides; In Vitro Techniques; Inosine Monophosphate; Kidney; Kinetics; Male; Nitrogen Isotopes; Perfusion; Rats; Rats, Inbred Strains; Reference Values

The rate of AMP deamination to IMP and NH4, by the action of AMP deaminase, is increased in vitro by acidosis and elevations in [AMP] and [ADP]. We evaluated the influence of acidosis on the activity of AMP deaminase in contracting muscle (5 Hz) by relating the time course of IMP and NH4 production to lactate-induced acidosis in low-oxidative, fast-twitch white (FTW) and high-oxidative, fast-twitch red (FTR) muscle of the rat. Cellular acidosis was modified by controlling lactic acid accumulation by regulating muscle blood flow and using trained animals. A significant activation of AMP deaminase occurred in both muscle types, but only at times when the estimated pH was 6.6 and below (lactate content 20 mu mol/g and above). Cellular acidosis, however, is not absolutely essential, since iodoacetic acid-blocked muscle lost 85-90% of its ATP to IMP during contractions. Thus cellular acidosis seems to be an important, but not the sole, factor activating AMP deaminase during contractions. Further, the influence of acidosis is probably different between fiber types, since the estimated free AMP and ADP contents, calculated from the creatine kinase and myokinase reactions, were different in the two fiber types. Most of the activation of AMP deaminase in FTR muscle could be attributed to a substrate effect of the increased free AMP content. In contrast, most of the activation of AMP deaminase in the FTW muscle was due to factors other than a substrate effect. These results suggest that cellular acidosis during intense contraction conditions is a major factor activating AMP deaminase, especially in the low-oxidative FTW muscle fiber type.

Topics: Acidosis; Adenine Nucleotides; AMP Deaminase; Animals; Electric Stimulation; Energy Metabolism; Inosine Monophosphate; Ischemia; Kinetics; Male; Muscle Contraction; Muscles; Nucleotide Deaminases; Rats; Rats, Inbred Strains