Phosphorylated metabolites and effects of amphotericin B and ouabain on phosphorylation state in amphibian cornea.
The phosphorylated metabolite content of endothelial denuded frog corneas and toad corneal epithelial cells was measured. The extract of frog corneas contained adenine, uridine and guanine nucleotides, in addition to various glycolytic intermediates, orthophosphate (Pi) and creatine phosphate. The relationship between the phosphorylation state: PS = [ATP]/[ADP] X [Pi] and the rate of active ion transport was studied in the toad cornea. This was considered by measuring the effects of 10(-5) M-amphotericin B and 10(-2) M-ouabain on the adenine nucleotide and Pi content of toad epithelial cells since these drugs selectively stimulate and inhibit active ion transport and metabolic rates, respectively. In order to calculate PS values, we derived and validated a relationship for converting content in terms of nmol/mg protein into concentration (mM). Incubation with amphotericin B in six different cell suspensions resulted in decreases of the ATP and ADP concentrations from 2.3 to 1.2 mM and 0.7 and 0.4 mM, respectively. The Pi concentration increased more than the ATP decrease from 6.2 to 12.2 mM. The average of these individual changes in each of the suspensions resulted in a significant decrease of the calculated PS from 599 +/- 226 to 342 +/- 127 M-1. However, ouabain increased the ATP concentration from 2.3 to 2.6 mM and decreased the ADP and Pi concentrations from 0.7 to 0.3 mM and 6.2 to 5.4 mM, respectively. The average of these individual changes in each of the suspensions resulted in a significant increase of the PS from 599 +/- 226 to 2975 +/- 1639 M-1. These results support the current notion that the PS is the feedback signal synchronizing metabolic rates with the energetic requirements of active Na and K transport.
D000227:Adenine Nucleotides / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000666:Amphotericin B / Q000494:pharmacology*; D000818:Animals; D001001:Anura; D001693:Biological Transport, Active / Q000187:drug effects; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology*; D010766:Phosphorylation; D014500:Uracil Nucleotides / Q000378:metabolism
D000227:Adenine Nucleotides; D005136:Eye Proteins; D006150:Guanine Nucleotides; D014500:Uracil Nucleotides; D010042:Ouabain; D000244:Adenosine Diphosphate; D000666:Amphotericin B; D000255:Adenosine Triphosphate; D004247:DNA