Spermatozoa undergo a poorly understood activation process induced by bicarbonate and mediated by cyclic adenosine 3',5'-monophosphate (cAMP). It has been assumed that bicarbonate mediates its effects through changes in intracellular pH or membrane potential; however, we demonstrate here that bicarbonate directly stimulates mammalian soluble adenylyl cyclase (sAC) activity in vivo and in vitro in a pH-independent manner. sAC is most similar to adenylyl cyclases from cyanobacteria, and bicarbonate regulation of cyclase activity is conserved in these early forms of life. sAC is also expressed in other bicarbonate-responsive tissues, which suggests that bicarbonate regulation of cAMP signaling plays a fundamental role in many biological systems.

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H ؉ ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.We recently identified bicarbonate-activated soluble adenylyl cyclase (sAC) 1 as a chemosensor mediating bicarbonate-dependent elevation of cAMP (1), defining a potential transduction pathway for cells to sense variations in bicarbonate, as well as the closely related parameters, pCO 2 and pH (1-3). sAC is distinct from transmembrane adenylyl cyclases. It is insensitive to regulation by forskolin or heterotrimeric G proteins (2) but is directly activated by bicarbonate ions. It does not have predicted transmembrane domains and is present in both soluble and particulate fractions of cellular extracts (4 -6). Mammalian sAC is similar to bicarbonate-regulated adenylyl cyclases present in cyanobacteria (1, 2), suggesting there may be a unifying mechanism for the bicarbonate regulation of cAMP signaling in many biological systems.sAC is highly expressed in spermatozoa (7) where it is proposed to mediate the bicarbonate-dependent cAMP elevation that precedes capacitation, hyperactivated motility, and acrosome reaction needed for fertilization (1). While spermatozoa mature and are stored along the epididymal lumen, they are kept in a quiescent state by an acidic pH of 6.5-6.8 and a low bicarbonate concentration of 2-7 mM (8). We have previously shown (9, 10) that a sub-population of epithelial cells, the so-called clear cells, are important players in the acidification capacity of the epididymis. Clear cells express high levels of the V-ATPase in their apical pole, and are responsible for the bulk of proton secretion in the vas deferens. Proton secretion by clear cells occurs in a chloride-independent but bicarbonate-dependent manner (11). Similarly to kidney intercalated cells, epididymal clear cells regulate their rate of proton secretion via V-ATPase recycling between intracellular vesicles and the apical plasma membrane (12). In these cells, as well a...

Intracellular targets of the ubiquitous second messenger cAMP are located at great distances from the most widely studied source of cAMP, the G protein responsive transmembrane adenylyl cyclases. We previously identified an alternative source of cAMP in mammalian cells lacking transmembrane spanning domains, the "soluble" adenylyl cyclase (sAC). We now demonstrate that sAC is distributed in specific subcellular compartments: mitochondria, centrioles, mitotic spindles, mid-bodies, and nuclei, all of which contain cAMP targets. Distribution at these intracellular sites proves that adenylyl cyclases are in close proximity to all cAMP effectors, suggesting a model in which local concentrations of cAMP are regulated by individual adenylyl cyclases targeted to specific microdomains throughout the cell.

Intermittent fasting has been demonstrated to protect against Alzheimer's disease (AD), however, the mechanism is unclear. Histone acetylation and lipoprotein lipase (LPL) are involved in AD progression. Importantly, LPL has been documented to be regulated by histone deacetylases (HDACs) inhibitors (increase histone acetylation level) in adipocyte and mesenchymal stem cells, or by fasting in adipose and muscle tissues. In brain, however, whether histone acetylation or fasting regulates LPL expression is unknown. This study was designed to demonstrate intermittent fasting may protect against AD through increasing β-hydroxybutyrate, a HDACs inhibitor, to regulate LPL. We also investigated microRNA-29a expression associating with regulation of LPL and histone acetylation. The results showed LPL mRNA expression was increased and microRNA-29a expression was decreased in the cerebral cortex of AD model mice (APP/PS1), which were alleviated by intermittent fasting. No significant differences were found in the total expression of LPL protein (brain-derived and located in capillary endothelial cells from peripheral tissues) in the cerebral cortex of APP/PS1 mice. Further study indicated that LPL located in capillary endothelial cells was decreased in the cerebral cortex of APP/PS1 mice, which was alleviated by intermittent fasting. LPL and microRNA-29a expression were separately increased and down-regulated in 2 μM Aβ25−35-exposed SH-SY5Y cells, but respectively decreased and up-regulated in 10 μM Aβ25−35-exposed cells, which were all reversed by β-hydroxybutyrate. The increase of HDAC2/3 expression and the decrease of acetylated H3K9 and H4K12 levels were alleviated in APP/PS1 mice by intermittent fasting treatment, as well in 2 or 10 μM Aβ25−35-exposed cells by β-hydroxybutyrate treatment. These findings above suggested the results from APP/PS1 mice were consistent with those from cells treated with 2 μM Aβ25−35. Interestingly, LPL expression was reduced (0.2-folds) and microRNA-29a expression was up-regulated (1.7-folds) in HDAC2-silenced cells, but respectively increased (1.3-folds) and down-regulated (0.8-folds) in HDAC3-silenced cells. Furthermore, LPL expression was decreased in cells treated with microRNA-29a mimic and increased with inhibitor treatment. In conclusion, intermittent fasting inhibits the increase of brain-derived LPL expression in APP/PS1 mice partly through β-hydroxybutyrate-mediated down-regulation of microRNA-29a expression. HDAC2/3 may be implicated in the effect of β-hydroxybutyrate on microRNA-29a expression.