PDE5 activity was determined as described in Figure 2 ,

17 Jul 2018 23:28

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4B ). This showed that LH increases PDE5 phosphorylation at the site known to increase the hydrolytic activity of the enzyme 46 , 47 PDE5 phosphorylation increased within 10 min of LH treatment of follicles and remained elevated for 4 h ( Fig. Correspondingly, both PDE1 and PDE5 inhibitors elevated basal cGMP in the follicle ( Supplemental Fig. Addition of the PDE1 inhibitor PF-04822163 43 to the assay mixture reduced the basal cGMP-hydrolyzing activity in the follicle lysates by ∼45%, but did not inhibit the increase in cGMP hydrolysis in response to LH ( Fig.

2B ). PDE5 activity was significantly increased by 10 min of LH treatment, and remained elevated for at least 60 min ( Fig. Subtraction of the Fildena-insensitive activity from the total activity illustrated the increase in PDE5 activity in response to LH ( Fig. LH treatment of the follicles for 30 min increased the total cGMP-hydrolytic activity ( Fig.

Consistent with this hypothesis, LH signaling activates protein kinase A 24 , 25 An LH-induced increase in PDE5 activity would complement the LH-induced decrease in NPR2 activity, serving to reduce cGMP in the follicle and restart meiosis. In preovulatory follicles, meiotic arrest is maintained by cGMP that is produced in the granulosa cells and diffuses through gap junctions into the oocyte 1 - 4 Within the oocyte, cGMP inhibits the cyclic nucleotide phosphodiesterase PDE3A, maintaining a high level of cAMP in the oocyte 1 , 2 , which acts on cell cycle proteins to prevent the progression of meiosis 5 , 6 LH, which activates a receptor that is coupled to Gs and other G-proteins 7 - 9 , induces a rapid reduction in follicle cGMP, resulting in diffusion of cGMP out of the oocyte 4 This relieves the inhibition of PDE3A in the oocyte, lowering cAMP content and allowing meiosis to resume. Both the phosphorylation and the increase in activity were promoted by elevating cAMP and opposed by inhibiting protein kinase A, supporting the hypothesis that LH activates PDE5 by stimulating its phosphorylation by protein kinase A. Inhibition of PDE5 activity partially suppressed LH-induced meiotic resumption as indicated by nuclear envelope breakdown, but inhibition of both PDE5 and PDE1 activities was needed to completely inhibit this response.







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