Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. Using FRET-based cyclic nucleotide sensors in follicleenclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte, where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from ~2 μM to ~80 nM at 1 hour after LH stimulation) and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from ~1 μM to ~40 nM) relieves the inhibition of PDE3A, increasing its activity by ~5-fold. This causes a decrease in oocyte cAMP (from ~700 nM to ~140 nM), leading to the resumption of meiosis.

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Abstract-Cyclic nucleotide phosphodiesterases regulate cAMP-mediated signaling by controlling intracellular cAMP content.The cAMP-hydrolyzing activity of several families of cyclic nucleotide phosphodiesterases found in human heart is regulated by cGMP. In the case of PDE2, this regulation primarily involves the allosteric stimulation of cAMP hydrolysis by cGMP. For PDE3, cGMP acts as a competitive inhibitor of cAMP hydrolysis. Several cGMP-mediated responses in cardiac cells, including a potentiation of Ca 2ϩ currents and a diminution of the responsiveness to ␤-adrenergic receptor agonists, have been shown to result from the effects of cGMP on cAMP hydrolysis. These effects appear to be dependent on the specific spatial distribution of the cGMP-generating and cAMP-hydrolyzing proteins, as well as on the intracellular concentrations of the two cyclic nucleotides. Gaining a more precise understanding of how these cross-talk mechanisms are individually regulated and coordinated is an important direction for future research. (Circ Res. 2007;100:1569-1578.) Key Words: phosphodiesterases Ⅲ signaling cross-talk Ⅲ cAMP Ⅲ cGMP Ⅲ compartmentalization T he second messengers cAMP and cGMP are important regulators of cardiac function. cAMP, which is generated by adenylyl cyclases (AC) on G protein-coupled receptor stimulation by catecholamines, regulates the strength and frequency of cardiac contraction and relaxation. The main downstream effector of cAMP is protein kinase A (PKA), 1 though cyclic nucleotide-gated ion channels and the exchange factor for Rap, Epac, 2 are also cAMP targets. cGMP, which is generated by guanylyl cyclases (GC) in response to nitric oxide (NO) and natriuretic peptides, modulates inotropy and metabolic responses 3 via the activation of its downstream effectors, protein kinase G (PKG) and cyclic nucleotide-gated channels. These two signaling pathways often exert opposing influences on cardiac function, 3 in part as a consequence of the opposing effects of PKA-and PKG-mediated phosphorylation on target proteins.A separate level of cross-talk between the cAMP and cGMP signaling pathways involves the activity of the cyclic nucleotide-degrading enzymes, phosphodiesterases (PDEs). In the heart, cGMP acts as a regulator of the activity of cAMPhydrolyzing PDEs, such that the intracellular concentration of cGMP can influence the intracellular concentration of cAMP.Original received November 11, 2006; revision received March 20, 2007; accepted March 27, 2007. From the Dulbecco Telethon Institute at the Venetian Institute of Molecular Medicine (M.Z.), Padova, Italy; and the Cardiology Section, Veterans Affairs Salt Lake City Health Care System (M.A.M.), and Departments of Internal Medicine (Cardiology) and Pharmacology, University of Utah School of Medicine, Salt Lake City, Utah.Correspondence The modulatory effects of cGMP on cAMP-hydrolyzing PDEs occur at nanomolar to micromolar concentrations of cGMP. This concentration range is comparable to that at which cGMP activates canonical targets such as PKG (K ...

All Mendelian hypertension syndromes described to date involve increased sodium reabsorption in the distal nephron. 5 The sole exception is autosomal-dominant hypertension with BDE (HTNB, OMIM #112410), first reported in a Turkish kindred. 2,6 HTNB was linked to chromosome 12p in six unrelated families. 2,7,8 The locus accounts for a ~50 mm Hg mean blood pressure difference at age 50 years. 2 The penetrance is 100% (Fig. 1a). Previously, we reported a rearrangement on chromosome 12p common to all families. 8,9 A linkage study in Chinese hypertensive families without BDE coincided with the HTNB locus, supporting relevance to essential hypertension. 10 Whole-genome sequencing of Turkish family members revealed a heterozygous missense mutation in PDE3A (Gene ID: 5139), a gene encoding a cGMP/cAMP phosphodiesterase with a prominent role in the heart, VSMC, oocytes and platelets. 11 Resequencing of all 48 affected persons in six unrelated families identified six independently clustered heterozygous missense mutations in exon 4 (Fig. 1a, b Supplementary Fig. 1).We detected none of the previously described chromosomal breakpoints on chromosome 12p12.2-12.1, perhaps due to high repetitive content in the breakpoint regions Fig. 2a-c). 4 A haplotype analysis identified a novel recombination that reduced the linkage interval and eliminated an inversion common to all affected individuals in the six families (Fig. 2c). 9 In contrast, the affected mother's haplotype showed co-segregation with the more severe brachydactyly phenotype.PDEs are involved during early stages of osteogenesis. 12 PDE4D mutations have been associated with severe brachydactyly in acrodysostosis. 13,14 In mice, Pde3a was expressed in the developing limbs, consistent with a role during chondrogenesis (Fig. 2d, Supplementary Fig. 3a, b). Chondrogenic downregulation of PTHLH encoding PTHrP was associated with BDE. 15 We also observed PTHLH downregulation in chondrogenically induced fibroblasts from affected persons (Fig. 2e, Supplementary Fig. 3c).We addressed the functional consequences of the identified PDE3A mutations in HeLa cells expressing the six mutations. Forskolin or L-arginine stimulated the adenylate or guanylate cyclases to enhance cellular cAMP or cGMP levels, respectively. 16,17 We detected significantly reduced cAMP levels, consistent with gain-of-function mutations with no change in cGMP levels for the PDE3A mutations ( Supplementary Fig. 4a, b). Three PDE3A isoforms, PDE3A1 (microsomal), PDE3A2 and PDE3A3 (microsomal and cytosolic), have been identified in human myocardium. 18,19 PDE3A3 does not contain the sequence harboring the detected mutations. The predominant isoform in VSMC is PDE3A2. 18,20 To directly elucidate the mutations' effects, we compared the Michaelis-Menten kinetics of cAMPhydrolytic activity for recombinant T445N FLAG-tagged PDE3A1 and PDE3A1-WT and the tagged A2 isoforms purified from transfected cells (Fig. 3a, b, Supplementary Fig. 4d-k). The T445N mutation increased the affinity of both enzyme's isoforms for cAM...