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Soluble Adenylyl Cyclase as an Evolutionarily Conserved Bicarbonate Sensor

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Science  28 Jul 2000:
Vol. 289, Issue 5479, pp. 625-628
DOI: 10.1126/science.289.5479.625

Abstract

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.

Ejaculated spermatozoa are not competent to fertilize an egg. They must first undergo a number of bicarbonate-induced processes, including the induction of hyperactivated motility, capacitation, and the acrosome reaction (1–3). Each of these processes is known to be cAMP-dependent, but they do not appear to involve the widely studied, hormone-responsive, transmembrane adenylyl cyclases (tmACs). We recently described the purification and cloning of a form of mammalian adenylyl cyclase, the sAC, that is structurally, molecularly, and biochemically distinct from the heterotrimeric guanosine triphosphate–binding protein (G protein)–regulated tmACs (4). The sAC cDNA encodes a 187-kD protein that is proteolytically processed to the mature 48-kD isoform purified from rat testes (4). Regulators of sAC activity have not yet been identified; tmAC modulators such as G proteins and forskolin do not affect sAC activity (4–6). Because sAC message is most abundantly expressed in male germ cells (4, 7) and its activity is distinct from that of tmACs, we tested whether sAC mediated the bicarbonate-induced cAMP increase in sperm.

Western blotting confirmed sAC's presence in bicarbonate-responsive spermatozoa. Anti-sAC antisera (8) detected the 48-kD mature form (4) and also detected higher molecular weight precursors in both rat testis and mouse epididymal sperm (Fig. 1B). Native sAC was also detected in other tissues known to regulate bicarbonate concentrations and reported to contain bicarbonate-stimulated AC activity (9), such as the kidney and the choroid plexus (Fig. 1B). These antisera specifically immunoprecipitated a bicarbonate-stimulated AC activity from the cytosol of rat testis (Fig. 1C) (10). The activity was not forskolin-responsive and is therefore unlikely to be caused by cross-reacting tmACs in the immunoprecipitate. Contrary to previous reports in which its in vitro activity required nonphysiologically relevant concentrations of Mn2+–adenosine triphosphate (ATP) (4, 11), sAC activity in these immunoprecipitates was measured in the presence of the more physiologically relevant substrate Mg2+-ATP. These data suggest that sAC is responsible for bicarbonate-stimulated cAMP accumulation in the testis and sperm and that bicarbonate may be acting directly on sAC enzymatic activity.

Figure 1

sAC is present in bicarbonate-sensing tissues. (A) Antisera to sAC specifically recognize heterologously expressed sAC protein in HiFive cells infected with recombinant baculoviruses expressing the indicated proteins (“empty” refers to empty baculovirus vector). The predicted sizes of heterologously expressed proteins are indicated on the right. (B) Anti-sAC Western blot of the testis (30 μg), sperm (5 μg), kidney (50 μg), and choroid plexus (50 μg). Different amounts of total protein were loaded to allow direct comparison in one exposure. The molecular weight of the predominant native sAC isoform is indicated on the right. (C) AC activity in immunoprecipitates from testis cytosol, using either preimmune serum or α-sAC antisera, was measured by radioimmunoassay (Amersham) in the absence of any additions (striped bar) or in the presence of 10 mM ATP, 10 mM MgCl2, and 40 mM NaHCO3 (black bar) or 100 μM forskolin (gray bar). Data are presented as picomoles of cAMP formed over 20 min, and values represent averages of duplicate determinations, with SDs indicated by error bars.

The effect of bicarbonate on sAC activity was tested in a stable HEK293 cell line expressing the full-length (sACfl) cDNA (HEK293/sACfl). Addition of NaHCO3 to the extracellular medium stimulated cAMP accumulation in HEK293/sACflcells but not in vector-transfected HEK293 cells (HEK293/V) (Fig. 2A) (12). The bicarbonate concentrations used in this experiment mimic the increase observed in sperm (from ≤5 mM in caudal epididymal sperm before ejaculation to ≥25 mM after ejaculation) (13). Bicarbonate increased cAMP production within minutes of its addition (14), concomitant with an observed elevation of intracellular pH indicating that bicarbonate had entered the cell (15). These data demonstrate that sAC can be activated by bicarbonate in a cellular context in the absence of any additional testis- or sperm-specific factors.

Figure 2

sAC is stimulated by bicarbonate. (A) Cellular cAMP accumulation was measured in stable cell lines expressing expression vector alone (diamonds) or sACfl (squares) at the indicated concentrations of NaHCO3 after growth for 24 hours in bicarbonate-free medium. Data are expressed as cAMP formed relative to total adenine nucleotides, and values represent averages of quadruplicate determinations with SDs indicated by error bars. (B) In vitro cyclase activity (measured in the presence of 5 mM MgCl2) in extracts from stable cell lines expressing empty expression vector, sACfl, or sACt in the presence (dark bars) or absence (light bars) of 50 mM NaHCO3. Data are expressed as picomoles of cAMP formed per minute per milligram of total cellular protein, and values represent averages of triplicate determinations, with SDs indicated by error bars.

To delineate the regions of sAC that mediate bicarbonate activation, we constructed an additional stable cell line (HEK293/sACt) expressing a catalytically active NH2-terminal truncation consisting almost exclusively of the two catalytic domains (sACt), which approximates the native 48-kD species (4). Bicarbonate also stimulated cAMP accumulation in HEK293/sACt cells (14), revealing that bicarbonate stimulation of sAC activity does not require the large COOH-terminal domain. Bicarbonate also activated heterologously expressed sAC in vitro. AC activity was stimulated in cellular lysates from HEK293/sACfl and HEK293/sACt cells (Fig. 2B), which is consistent with the idea that the enzyme is directly modulated by bicarbonate ions.

To demonstrate that bicarbonate acts directly on sAC and to exclude the possibility of accessory factors mediating activation in preparations from testis (Fig. 1C) and stable cell lines (Fig. 2), we purified recombinant sACt protein (16). Purified enzyme was stimulated more than sevenfold (Fig. 3A) with a median effective concentration (EC50) (25.4 ± 7.6 mM) within the physiologically relevant bicarbonate concentration in mammalian serum (22 to 26 mM) (17, 18). Presumably, this direct activation of sAC accounts for the observed intracellular increase in cAMP generation in sAC-expressing cell lines (Fig. 2A).

Figure 3

Bicarbonate activation of sAC is direct, specific, and pH-independent. (A) Purified sACtwas assayed in the presence of the indicated concentrations of NaHCO3 with 10 mM ATP and 40 mM MgCl2. Data are expressed as nanomoles of cAMP formed per minute per milligram of protein, and values are averages of triplicate determinations. (B) Purified sACt was assayed as in (A) at the indicated final pHs (buffered by tris-HCl) in the presence (circles) or absence (squares) of 40 mM NaHCO3. Best-fit lines were generated by means of linear regression analysis. (C) Engineered soluble tmAC was constructed and purified as described (19) and assayed in the presence of MgCl2 alone (basal) or with 50 mM NaHCO3 or 100 μM forskolin. Data are presented as picomoles of cAMP formed per minute per milligram of protein, and values represent triplicate determinations with SDs indicated by error bars.

Bicarbonate stimulation was not due to altered pH, because both Mg2+-ATP alone and bicarbonate-stimulated sAC activities were completely insensitive to pH changes over the range 7.0 to 8.5 (Fig. 3B). sAC activity was stimulated equally well by NaHCO3 or KHCO3, and we were able to mimic the stimulatory effects of NaHCO3 using bisulfite ion (Na2SO3 or NaHSO3), which structurally resembles bicarbonate, but not with dissimilar ions, such as chloride (NaCl), sulfate (Na2SO4), or phosphate (Na2HPO4) (Table 1). These data exclude Na+ ion and simple alterations of ionic strength as regulators of sAC activity, and they indicate that bicarbonate, as opposed to CO2, directly binds to and activates sAC in a pH-independent manner. However, because carbon dioxide is in equilibrium with bicarbonate, sAC and the cAMP signaling pathway may also indirectly monitor in vivo levels of carbon dioxide.

Table 1

Activation of sAC by various salts.

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Among mammalian ACs, sAC appears to be the only form regulated by bicarbonate ions. HEK293/V cells, which express endogenous tmACs, are unaffected by the addition of bicarbonate (Fig. 2). Even when submaximally stimulated by forskolin, tmAC activity was insensitive to bicarbonate (14). Finally, although a purified, engineered, soluble form of tmAC [type V (19)] was fully responsive to forskolin, it was completely insensitive to bicarbonate addition (Fig. 3C). Therefore, bicarbonate stimulation is not a general feature of all ACs, and mammalian cells possess two independently regulated cAMP signal transduction systems.

The two catalytic domains of sAC (C1 and C2) (4) more closely resemble the active portions of cyanobacterial ACs than those from mammalian tmACs (Fig. 4A). It has been hypothesized that cyanobacteria (blue-green algae) were the predominant form of life in the carbon dioxide–rich, pre-Cambrian environment (20). They are thought to be responsible for the photosynthesis that transformed early Earth's carbon dioxide–rich atmosphere into an oxygen atmosphere (20). cAMP is known to regulate respiration in cyanobacteria (21), but there is no known molecule that modulates their AC activity. The similarity between sAC and cyanobacterial ACs prompted us to examine whether bicarbonate regulation of cAMP signaling is conserved in cyanobacteria. The AC activity of purified Spirulina platensis CyaC was stimulated 2.5-fold by the presence of bicarbonate ions (Fig. 4B). Similar to mammalian sAC, bicarbonate-stimulated cyanobacterial CyaC with an EC50 of 18.8 ± 1.6 mM and bicarbonate regulation was pH-independent (14). These data demonstrate that cyanobacterial ACs can also serve as bicarbonate sensors and that bicarbonate regulation of AC activity is conserved across phyla separated by hundreds of millions, if not billions, of years.

Figure 4

Bicarbonate activates cyanobacterial AC. (A) Phylogenetic relationship between catalytic domains from a variety of ACs aligned with the use of CLUSTALW (DNA*), represented as an unrooted dendogram constructed with PROTPARS (PHYLIP 3.5) (22). Numbers represent bootstrap confidence values. Accession numbers for the aligned amino acid sequences are as follows: sAC [rat sAC: AAD04035], tmAC1 [bovine Type I: AAA79957], tmAC2 [rat Type II: AAA40682], tmAC5 [rat Type V: Q04400], tmAC9 [mouse Type IX: CAA03415], D.d. AcrA [Dictyostelium discoideumAcrA: AAD50121], A.sp. (Anabaena spirulina) cyaA [BAA13997], A.sp. cyaB1 [BAA13998], A.sp. cyaB2 [BAA13999], A.sp. cyaC [BAA14000], A.sp. cyaD [BAA14001], S.pl. (Spirulina platensis) CyaA [BAA22996], S.pl. CyaC [BAA22997], Syn. (Synechocystis sp.) CyaA1 [BAA16969], and Syn. CyaA2 [BAA17880]. (B) Spirulina platensis CyaC, expressed and purified as previously described (23), was assayed in the presence of the indicated concentrations of NaHCO3 with 100 μM ATP and 5 mM MnCl2(23, 24). Data are expressed as picomoles of cAMP formed per minute per milligram of protein, and values are averages of triplicate determinations.

We have demonstrated that the physiologically ubiquitous ion bicarbonate stimulates the production of a second messenger molecule, cAMP, by direct modulation of enzymatic activity. Our data suggest that sAC is the chemosensor mediating bicarbonate's effects during sperm activation, and they define sAC as a potential target for male contraceptives. Furthermore, the expression of mammalian sAC in other bicarbonate-responsive tissues and the evolutionary conservation of bicarbonate-mediated cAMP generation suggest that this signal transduction pathway mediates a wide variety of biological processes.

  • * These authors contributed equally to this work.

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