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.

Mammals have nine differentially regulated isoforms of G protein-responsive transmembrane-spanning adenylyl cyclases. We now describe the existence of a distinct class of mammalian adenylyl cyclase that is soluble and insensitive to G protein or Forskolin regulation. Northern analysis indicates the gene encoding soluble adenylyl cyclase (sAC) is preferentially expressed in testis. As purified from rat testis cytosol, the active form of sAC appears to be a fragment derived from the full-length protein, suggesting a proteolytic mechanism for sAC activation. The two presumptive catalytic domains of sAC are closely related to cyanobacterial adenylyl cyclases, providing an evolutionary link between bacterial and mammalian signaling molecules.Adenylyl cyclase (AC) is the effector molecule of one of the most widely used signal transduction pathways. Its product, cAMP, mediates cellular responses to nutritional conditions and extracellular signals in organisms from prokaryotes to higher eukaryotes. In metazoans, a seemingly ubiquitous membrane-associated AC activity is encoded by a family of transmembrane adenylyl cyclases (tmACs) that mediate cellular responses to external stimuli. In mammals, nine distinct tmAC genes differing in their patterns of expression and regulatory properties have thus far been identified. Their catalytic activities are differentially regulated by G proteins and other signaling molecules in response to stimuli such as hormones and neurotransmitters (1, 2).In addition, another type of AC activity had been described in mammals. A soluble enzymatic activity was detected in cytosolic extracts from mammalian testis (3). Soluble AC activity appeared to be biochemically and chromatographically different from the tmACs and soluble guanylyl cyclases previously described in testis (4-6). Unlike the known tmACs, its biochemical activity depended on the divalent cation Mn 2ϩ (3), was insensitive to G protein regulation (6), and displayed approximately 10-fold lower affinity for substrate, ATP (K m Ϸ1 mM) (4, 7, 8) than the tmACs (K m Ϸ100 M) (9). Based on these studies, this soluble form of AC was predicted to be molecularly distinct from tmACs (8, 10).A soluble form of AC would define a novel means for generating cAMP and would imply that the second messenger could be generated at a distance from the membrane, closer to its required site of action. However, the molecular evidence confirming that soluble AC represents a distinct form of AC had been lacking. We now describe purification, molecular cloning, and functional expression of the cDNA encoding the soluble form of AC (sAC). The full-length cDNA predicts a protein of 187 kDa, whereas the catalytically active purified form of the enzyme is only 48 kDa, suggesting a proteolytic mechanism of activation for sAC. Two distinct regions within the catalytically active portion of sAC are very similar to catalytic portions of ACs from cyanobacteria and myxobacteria, providing a link between bacterial and mammalian signaling systems. MATERIALS AND METH...

Mitochondria constantly respond to changes in substrate availability and energy utilization to maintain cellular ATP supplies, and at the same time control reactive oxygen radical (ROS) production. Reversible phosphorylation of mitochondrial proteins has been proposed to play a fundamental role in metabolic homeostasis, but very little is known about the signalling pathways involved. We show here that Protein Kinase A (PKA) regulates ATP production by phosphorylation of mitochondrial proteins, including subunits of cytochrome c oxidase. The cyclic AMP (cAMP) which activates mitochondrial PKA does not originate from cytoplasmic sources but is generated within mitochondria by the carbon dioxide/bicarbonate-regulated soluble adenylyl cyclase (sAC) in response to metabolically generated carbon dioxide. We demonstrate for the first time the existence of a CO2-HCO3−-sAC-cAMP-PKA (mito-sAC) signalling cascade wholly contained within mitochondria, which serves as a metabolic sensor modulating ATP generation and ROS production in response to nutrient availability.

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.

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