ラット腎cyclic nucleotide phosphodiesteraseに及ぼす各種ホルモンの影響
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Kidney function is regulated by several hormones which act through adenylate cyclasecyclic AMP system. The present study was undertaken to investigate cyclic AMP- and cyclic GMP-phosphodiesterase (cAMP-PDE and cGMP-PDE respectively) activities in the rat kidney, and also the effect of several hormones affecting the kidney function on these enzyme activities in vitro. <BR>Rat kidneys were separated into cortex and medulla. These were homogenized in 50 mM Tris-HC1 buffer, pH 7.5, containing 0.32 M sucrose and fractionated by centrifugation. PDE activity was measured in all fractions, using the two-step assay system. A low substrate concentration (0.5 μM) was used, unless otherwise stated. <BR>Substantial activity was present in all of the fractions and most of the activity existed in the soluble fraction (105000 × g supernatant). Cyclic GMP-PDE activity was dominant in both cortex and medulla. The rat kidney contained two forms of cAMP-PDE, one of which had a Km of 2.0 × 10<SUP>-4</SUP>M and another which had a low Km of 2.5 × 10<SUP>-5</SUP>M, and one form of cGMP-PDE with a Km of 2.5 × 10<SUP>-5</SUP>M. These cAMP-PDE and cGMP-PDE were purified by Sepharose-6B column chromatography. Cyclic AMP-PDE activity was found in a broad area associated with two peaks and cGMP-PDE activity had one peak corresponding to the same peak as the hgih molecular weight cAMP-PDE. Calmodulin was eluted after the peak of cGMP-PDE activity. Both cAMP-PDE and cGMP-PDE activities were inhibited by calcium ion at a concentration of more than 5.0 × 10<SUP>-4</SUP>M. Cyclic GMP-PDE activity was not activated by calmodulin in the presence of enough calcium ion.<BR>The effect of 1α, 25(OH)<SUB>2</SUB> Vit D<SUB>3</SUB>, parathyroid hormone (PTH), antidiuretic hormone (ADH), calcitonin (CT), angiotensin II, and trichrolomethiazide on the partially purified cAMP-PDE and cGMP-PDE activities were examined. 1α, 25(OH)<SUB>2</SUB> Vit D<SUB>3</SUB> activated cAMP-PDE activity and did not affect cGMP-PDE activity. The concentrations of 1α, 25(OH)<SUB>2</SUB> Vit D<SUB>3</SUB> producing 50% activation of cAMP-PDE activity were 5.0 × 10<SUP>-11</SUP>M (cortex) and 6.7 × 10<SUP>-10</SUP>M (medulla). CT and ADH inhibited both cAMP-PDE and cGMP-PDE activities. The concentrations of CT producing 50% inhibition of cAMP-PDE activity were 4.0 ×10<SUP>-5</SUP>M (cortex) and 3.3 × 10<SUP>-7</SUP>M (medulla), and those of cGMP-PDE activity were 1.0 × 10<SUP>-5</SUP>M (cortex) and 1.0 × 10<SUP>-4</SUP>M (medulla). Concerning ADH, the concentrations required for 50% inhibition of cAMP-PDE activity were 5.3 × 10<SUP>-6</SUP>M (cortex) and about 1.0 × 10<SUP>-3</SUP>M (medulla), and those of cGMP-PDE activity were 5.3 × 10<SUP>-3</SUP>M (cortex) and 5.3 × 10<SUP>-8</SUP>M (medulla). CT inhibited cAMP-PDE activity more effectively than cGMP-PDE activity, whereas the inhibition by ADH was vice sera. PTH, angiotensin II, and trichrolomethiazide did not affect these enzyme activities. <BR>The data described here imply that these hormones may control kidney function through cyclic nucleotide metabolism by affecting cAMP-PDE and/or cGMP-PDE activity.
- 一般社団法人 日本内分泌学会の論文
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- ラット腎cyclic nucleotide phosphodiesteraseに及ぼす各種ホルモンの影響