HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 18, 15922-15926, May 2, 2003

This Article Abstract Full Text (PDF) All Versions of this Article: 278/18/15922    most recent M212475200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Litvin, T. N. Articles by Levin, L. R. Search for Related Content PubMed PubMed Citation Articles by Litvin, T. N. Articles by Levin, L. R. Social Bookmarking                      What's this?

Kinetic Properties of "Soluble" Adenylyl Cyclase

SYNERGISM BETWEEN CALCIUM AND BICARBONATE^*

Tatiana N. Litvin, Margarita Kamenetsky, Alla Zarifyan, Jochen Buck, and Lonny R. Levin

From the Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021

Received for publication, December 9, 2002, and in revised form, February 13, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

"Soluble" adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V_max of the enzyme with little effect on the apparent K_m for substrate, ATP-Mg²⁺. Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparent K_m for ATP-Mg²⁺. Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC; therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Two types of adenylyl cyclase (AC)¹ are ubiquitously expressed in mammalian cells, a well characterized gene family of transmembrane ACs (tmACs) and the recently discovered "soluble" AC (sAC). The tmACs are plasma membrane bound, and their activities are regulated by G proteins in response to extracellular stimuli such as neurotransmitters and hormones (reviewed in Ref. 1). In contrast, sAC is associated with various intracellular organelles, including mitochondria, centrioles, mitotic spindle, mid-bodies, and nuclei (2). sAC activity is modulated by bicarbonate (3) and, as shown in this report, by Ca²⁺; regulation by these intracellular signaling molecules suggests that sAC mediates cAMP-dependent responses to intrinsic cellular changes (4, 5).

The catalytic mechanism of tmACs has been determined from biochemical and crystallographic studies. tmACs convert ATP to cAMP using two-metal catalysis where one ion acts as a free metal and the other coordinates ATP in the active site (6, 7). Its activators, G_s subunit or forskolin, stimulate tmACs by allosteric modulation of the active site (8, 9). More than 25 years ago, when soluble AC activity was first discovered, it was predicted to be molecularly distinct from tmACs because its activity appeared to be dependent on the presence of the divalent cation, Mn²⁺, and it was insensitive to forskolin and G protein regulation (10-12). These differential properties enabled purification (13) and cloning of sAC from rat testis (14). The sAC gene is indeed molecularly distinct from tmACs; it possesses no transmembrane domains, and its catalytic domains are more closely related to those of cyanobacterial ACs than to those from other eukaryotic ACs. The purified soluble AC exhibited ~10-fold lower affinity for substrate ATP relative to tmACs (tmAC K_m for ATP-Mn²⁺ is ~ 100 µM, whereas purified rat testis sAC K_m for ATP-Mn²⁺ is ~ 1 mM), and the activity of the heterologously expressed, cloned sAC gene product is insensitive to forskolin or G proteins (14).

Soluble AC, as the predominant, if not only, source of cAMP in sperm, was predicted to be responsible for the cAMP changes induced by seminal and oviductal fluids (and mimicked by in vitro fertilization (IVF) media) necessary for fertilization of an egg, capacitation, hyperactivated motility, and acrosome reaction (15-18). Two essential components of defined IVF media are bicarbonate and calcium, and we previously demonstrated that sAC is directly stimulated by physiological levels of the bicarbonate anion (3). The role of calcium in IVF media is less clear because of contradicting reports detailing Ca²⁺ modulation of sperm cyclase (19-21). We performed kinetic analyses on purified recombinant 48-kDa truncated human sAC protein (sAC_t) fused to GST. This truncated protein corresponds to a splice variant of the sAC gene (22) that consists almost exclusively of the sAC catalytic domains and corresponds to the native isoform originally purified from testis cytosol (14). We identify a synergistic interaction between bicarbonate and calcium ions, where bicarbonate functions to increase the V_max of the enzyme whereas calcium increases its affinity for substrate ATP-Mg²⁺.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- ATP, chlorpromazine, and LaCl₃ were purchased from Sigma; all tissue culture reagents were from Invitrogen and [³²P]ATP and [³H]cAMP were from PerkinElmer Life Sciences.

Cloning of Human GST·sAC-- Human sAC_t was subcloned using gateway cloning technology (Invitrogen) into a baculovirus expression vector utilizing the polyhedron promoter to generate an N-terminal glutathione S-transferase (GST) fusion protein. Recombinant GST·human sAC_t-expressing baculovirus was produced in adherent SF9 cells (Bac-to-Bac baculovirus expression systems, Invitrogen), and identity of the resultant fusion protein was confirmed by Western blotting and enzymatic activity.

Expression and Purification of GST·sAC Fusion Protein-- Hi-Five cells, grown to a density of ~1.0 × 10⁶ cells per ml, were infected with GST·human sAC_t baculovirus. After 48 h, cells were pelleted, resuspended in lysis buffer (phosphate-buffered saline with 1 mM EDTA, pH 7.4, 1 mM dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), and lysed by sonication. The cell lysate was cleared by centrifugation at 17,500 × g for 60 min at 4 °C, and the supernatant was applied to a glutathione-Sepharose 4B column (Amersham Biosciences). The GST·human sAC_t fusion protein was eluted with glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0, 10 µg/ml aprotinin, 10 µg/ml leupeptin). GST·human sAC_t was further purified by gel filtration over Superdex 200 HR 10/30 column (Amersham Biosciences); sAC-containing fractions were stored in 50% glycerol at 20 °C. Coomassie Blue-stained SDS-PAGE reveals one predominant band corresponding to GST·human sAC_t and a minor contaminant corresponding to GST alone (Fig. 1, inset). Multiple independent assays comparing the GST·sAC_t fusion protein with cleaved and re-purified sAC_t confirmed that the GST fusion does not affect enzymatic activity or kinetic parameters of sAC (data not shown).

Cyclase Assay-- Cyclase assays were performed in 100 µl of total reaction volume using ~100 ng of purified GST·human sAC_t fusion protein in the presence of 50 mM Tris-HCl, pH 7.5, substrate [-³²P]ATP, and either MnCl₂, MgCl₂, and/or CaCl₂ as indicated. Reactions were incubated at 30 °C for 30 min unless otherwise noted and were stopped by adding 200 µl of 2% SDS. [³²P]cAMP generated by the reaction was recovered using the two-column method (23, 24). For kinetic analysis, sAC activity was assayed as a function of varying ATP-Mn²⁺ or ATP-Mg²⁺ in the presence of excess MnCl₂, MgCl₂, or CaCl₂. ATP was preincubated with MnCl₂ or MgCl₂, and serial dilutions were prepared; assays were started by addition of sAC protein. Kinetic analyses were performed using the program EnzymeKinetics v 1.11 (Trinity Software, Plymouth, NH).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ATP-Mn²⁺-dependent Activity of sAC-- Adenylyl cyclases require a divalent cation for catalytic activity. Historically, soluble adenylyl cyclase activity found in testis has been assayed in the presence of Mn²⁺ (10); little or no in vitro activity was detected when Mg²⁺, Ca²⁺, or Co²⁺ was used as the sole divalent (19, 25, 26). The K_m for ATP-Mn²⁺ of soluble AC activity from testis cytosol has been reported to be 1-2 mM (11, 21, 27). We now demonstrate that recombinant human sAC has similar activity, displaying a K_m of 0.8 mM ATP-Mn²⁺ (Fig. 1).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Activity of sAC in the presence of ATP-Mn2+. Soluble adenylyl cyclase activity was measured as a function of substrate ATP-Mn2+ in the presence of excess MnCl2 (10 mM) for 5 min. Km = 0.8 mM ATP-Mn2+ was determined using non-linear regression analysis. Graph is representative of three independent experiments. Inset, Coomassie Blue-stained 10% SDS-PAGE demonstrating the purity of GST·human sAC fusion protein used in these studies.

Bicarbonate Releases Substrate Inhibition by ATP-Mg²⁺ and Increases the V_max of sAC-- We recently described direct stimulation of rat sAC by the bicarbonate anion (3). Bicarbonate activation of sAC is thought to be at the center of fertilization-related processes that occur in all mammalian sperm, and we now confirm that purified human sAC is also stimulated by bicarbonate (Fig. 2A). Similar to rat sAC, in the presence of 10 mM ATP human sAC was stimulated up to 30-fold with a half-maximal effect (EC₅₀) of ~11 mM NaHCO₃. Bicarbonate stimulates sAC activity in the presence of magnesium, and because manganese is not found at the millimolar concentrations necessary to support sAC activity inside cells, magnesium represents the more physiologically relevant cation. Therefore, all subsequent experiments were performed using Mg²⁺-ATP as substrate.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Bicarbonate relieves ATP-Mg2+ substrate inhibition and increases enzyme velocity. A, human sAC activity was assayed in the presence of indicated concentrations of NaHCO3 with 10 mM ATP and 40 mM MgCl2. Values represent averages of triplicate determinations, with error bars indicating S.D. from the mean. B, sAC activity was measured as a function of substrate ATP-Mg2+ for 30 min in the presence of excess MgCl2 (20 mM) and increasing concentrations of NaHCO3, () 0 mM HCO; () 15 mM HCO; () 50 mM HCO. All determinations are representative of at least two independent experiments. Kinetic curves were generated by the EnzymeKinetics computer program using data points not reflecting substrate inhibition. C, sAC activity was measured as a function of substrate ATP-Mg2+ for 30 min in the presence of excess MgCl2 (80 mM) and NaHCO3 (80 mM).

In the absence of bicarbonate, activity of recombinant human sAC was inhibited at high ATP-Mg²⁺ concentrations. This substrate inhibition was relieved by the addition of bicarbonate (Fig. 2B). At 0 mM NaHCO₃, the onset of inhibition is at [ATP-Mg²⁺] > 6 mM; at 15 mM NaHCO₃ the onset is shifted to [ATP-Mg²⁺] > 13 mM and, at 50 mM NaHCO₃, substrate inhibition is virtually abated. The data indicate that, in addition to relieving the inhibition observed at high substrate concentrations, bicarbonate was not altering the apparent K_m for ATP-Mg²⁺; rather it was increasing the V_max of sAC (Fig. 2B). These 2-fold effects contribute to the observed 30-fold stimulation of activity (Fig. 2A). Similar effects were observed with purified recombinant rat sAC; bicarbonate increased the V_max of the enzyme and abated substrate inhibition (data not shown).

Previously published studies of crude soluble AC activity revealed an elevated K_m for ATP-Mg²⁺ (12-16 mM) relative to ATP-Mn²⁺ (1-2 mM) (21). We were unable to determine a true K_m for ATP-Mg²⁺ in the presence of NaHCO₃ because even at the highest NaHCO₃ (80 mM) and substrate (up to 30 mM ATP-Mg²⁺) concentrations used, sAC activity did not plateau (Fig. 2C). We could only conclude from non-linear regression analysis and Eadie-Hofstee plots that the apparent K_m for ATP-Mg²⁺ was greater than 10 mM, consistent with published reports using crude soluble AC activity (21).

Calcium Synergizes with Bicarbonate and Activates sAC by Decreasing Its K_m for ATP-Mg²⁺-- Like bicarbonate, CaCl₂ (1.7-3 mM) is an essential component of in vitro fertilization media, and Ca²⁺ has been implicated along with bicarbonate in activation of sperm cyclases (12, 15, 16, 19). We added CaCl₂ to bicarbonate-stimulated sAC and found enzymatic activity increased synergistically (Fig. 3A). CaCl₂ activation was dose-dependent with an EC₅₀ ~750 µM (Fig. 3B). Interestingly, the dose response to NaHCO₃ was unaffected by calcium; the EC₅₀ for NaHCO₃ remained ~11 mM in the presence or absence of CaCl₂ (Fig. 3C).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Synergistic activation of adenylyl cyclase by CaCl2 and NaHCO3. A, sAC activity was assayed in the presence of CaCl2 and/or NaHCO3 as indicated. All reactions contained 2.5 mM ATP and 5 mM MgCl2. Values represent averages of duplicate determinations and are representative of at least two independent experiments. B, sAC activity was assayed as a function of increasing CaCl2 concentration in the presence of 2.5 mM ATP, 5 mM MgCl2, and 40 mM NaHCO3. C, sAC activity was assayed as in Fig. 2A in the presence () or absence () of 10 mM CaCl2. D, sAC activity was assayed as a function of substrate ATP-Mg2+ for 30 min in the presence of excess CaCl2 (10 mM) and NaHCO3 (40 mM). Km = 0.9 mM for ATP-Mg2+ was determined by non-linear regression analysis. E, sAC activity was assayed as a function of substrate [ATP-Mg2+] for 30 min in the absence of any added modulator () or in the presence of 10 mM CaCl2 (), 40 mM NaHCO3 (), or 10 mM CaCl2 and 40 mM NaHCO3 (). Kinetic curves were generated by the EnzymeKinetics computer program using data points not reflecting substrate inhibition.

As shown above, bicarbonate stimulated sAC activity by alleviating ATP inhibition and by increasing V_max with little effect on apparent K_m. In contrast, kinetic analysis revealed that calcium had no effect on ATP inhibition and little effect on V_max (Fig. 3E) but stimulated sAC activity by decreasing its apparent K_m for ATP-Mg²⁺ (Fig. 3D). Addition of CaCl₂ causes a dramatic shift in the apparent K_m of sAC for ATP-Mg²⁺ from greater than 10 mM (Fig. 2B) to less than 1 mM (Fig. 3D). Overlaying the kinetic curves (Fig. 3E) illustrates how the effects of NaHCO₃ and CaCl₂ differ; NaHCO₃ activates sAC by increasing V_max, whereas CaCl₂ increases its affinity for substrate.

The Calcium Effect on sAC Is Direct and Independent of Calmodulin-- Coomassie Blue staining of purified human sAC did not reveal any substantial contaminating proteins (Fig. 1, inset), suggesting that calmodulin was not mediating the effect of calcium on sAC. To confirm that calmodulin was not involved, we found that adding exogenous bovine calmodulin did not have any affect on Ca²⁺-dependent sAC activity (data not shown) and inhibiting any potential calmodulin contamination using two independent calmodulin inhibitors, chlorpromazine or LaCl₃, had no effect on calcium-stimulated sAC activity (Fig. 4). Therefore, we conclude that the effect of CaCl₂ on sAC activity is because of direct binding of Ca²⁺, making sAC the first mammalian adenylyl cyclase to be stimulated by calcium directly. Specific tmAC isoforms are known to be stimulated by Ca²⁺, but they require calmodulin for the modulatory effect (23, 28-34).

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Effect of calmodulin inhibitors. sAC activity was measured in the presence of 2.5 mM ATP, 5 mM CaCl2, 5 mM MgCl2, and 40 mM NaHCO3 and the indicated concentrations of chlorpromazine () or LaCl3 (). Data points represent averages of duplicate determinations and are representative of at least two independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Soluble AC activity was first characterized in sperm and testis in the presence of MnCl₂ (10). Activity had been described in the presence of other divalents, such as Co²⁺, Ca²⁺, and Mg²⁺, but Mn²⁺-dependent activity was always significantly higher (19, 25, 26). We purified the 48-kDa isoform of human sAC fused to GST and confirmed that its K_m for ATP-Mn²⁺ (~0.8 mM) was indistinguishable from the value obtained for sAC activity purified from rat testis (14) and matched the values reported for soluble cAMP-producing activity in testis and sperm from a variety of mammals (11, 21, 27). These data confirm that the cloned sAC gene is responsible for the activities described in testis and sperm, and the kinetic parameters determined here correlate well with values established using native enzyme. However, it should be remembered that the kinetic analyses described in this study were performed on a heterologously expressed and purified enzyme; the in vivo properties of native sAC may differ because of post-translational modifications or interactions with regulatory proteins.

Soluble AC appears to be ubiquitously expressed (2, 35), and cells throughout the body do not possess concentrations of Mn²⁺ necessary to support sAC activity. We recently demonstrated that the ATP-Mg²⁺-dependent activity of purified rat sAC was stimulated by bicarbonate. Among mammalian adenylyl cyclases, bicarbonate regulation was unique to sAC; tmACs were unaffected by NaHCO₃ addition (3). Here we demonstrate that the human ortholog of sAC is also responsive to bicarbonate stimulation. The half-maximal effect (~11 mM NaHCO₃) is slightly lower for human sAC than the EC₅₀ reported (25 mM NaHCO₃) for rat sAC (3). Although both EC₅₀ for bicarbonate are within the physiological range (24-26 mM in most extracellular fluids and 10-15 mM inside cells), the difference might reflect species variation, or it could reflect differences between the two preparations; rat sAC was purified as a His₆·sAC fusion protein (3), whereas this study uses a GST fusion protein.

Bicarbonate can increase sAC activity in two ways; besides increasing enzyme velocity, bicarbonate also relieves the substrate inhibition observed at high ATP-Mg²⁺ concentrations. Therefore, the low (<5 mM) bicarbonate concentration in the epididymis (36, 37), where sperm are stored awaiting ejaculation, would further limit sAC activity in resting spermatozoa by allowing substrate inhibition. Upon ejaculation, the higher HCO (25 mM) found in seminal and prostatic fluids (which induces sperm motility and initiates capacitation) would lead to large, rapid increases in cAMP because of the increased V_max of sAC and relief from substrate inhibition.

Calcium (1.7-3 mM) is required in IVF media for capacitation, hyperactivated motility, and the acrosome reaction in sperm (15, 38). It is thought that Ca²⁺ is able to enter sperm via voltage-dependent and cyclic nucleotide gated calcium channels (39) as well as the putative cation channel CatSper (40). Previous reports demonstrated that detergent-dispersed adenylyl cyclase from guinea pig sperm membranes was activated by CaCl₂ (0.1-1 mM) in the presence of 5 mM MgCl₂ (19), and adenylyl cyclase from human epididymal sperm membranes was activated by 50 mM CaCl₂ and 50 mM NaHCO₃ (12). However, the molecular identity of the AC in these preparations remained unclear. In this report we confirm that calcium directly stimulates sAC activity and that calcium functions independently from calmodulin to increase the affinity of sAC for its substrate ATP-Mg²⁺.

Regulation by calcium, which is capable of supporting catalytic activity in the absence of Mg²⁺ (19, 25, 26), suggests that sAC, like tmACs, utilizes two metals in its active site; however, the active center of sAC would function best with different metals. It is possible that Ca²⁺, which lowers the apparent K_m for substrate ATP-Mg²⁺, would be better at coordinating ATP, while Mg²⁺ would serve as the catalytic metal.

The EC₅₀ for CaCl₂ stimulation appears to be high (~750 µM). However, it is important to keep in mind that CaCl₂ concentrations reported in this study do not reflect free Ca²⁺ concentrations. sAC activity would be responsive to the transiently elevated Ca²⁺ concentrations found during acrosome reaction and sperm motility, or sAC could be located near the pore of Ca²⁺ channels, such as the cyclic nucleotide gated ion channels (39), where it could mediate the cAMP regulation of channel opening. A microdomain consisting of sAC and cyclic nucleotide gated calcium channels could explain observed cAMP/calcium oscillations in cells (as proposed in Ref. 33). Because of the synergy between calcium and bicarbonate, even small intracellular changes in calcium or subtle changes in intracellular pH and/or carbon dioxide, which will be in equilibrium with bicarbonate, will result in significant changes of cellular cAMP.

	ACKNOWLEDGEMENTS

We thank Drs. Yanqiu Chen, Carmen Dessauer, Randi Silver, and Donald Fischman and members of the Levin/Buck laboratory for constructive advice and critical reading of the manuscript, Dr. Mime Kobayashi for cDNA preparation, and two anonymous reviewers for improvements to the manuscript.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Training Grant DA07274 (to T. N. L.), NIH Grants GM62328 and HD42060 (to J. B.) and HD38722 (to L. R. L.), and by the Ellison Medical Foundation (to J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 212-746-6274; Fax: 212-746-6241; E-mail: jobuck@med.cornell.edu.

Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M212475200

	ABBREVIATIONS

The abbreviations used are: AC, adenylyl cyclase; sAC, soluble AC; sAC_t, truncated isoform of sAC; tmAC, transmembrane AC; IVF, in vitro fertilization media; GST, glutathione S-transferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				L. S. Ramos, J. H. Zippin, M. Kamenetsky, J. Buck, and L. R. Levin Glucose and GLP-1 Stimulate cAMP Production via Distinct Adenylyl Cyclases in INS-1E Insulinoma Cells J. Gen. Physiol., August 25, 2008; 132(3): 329 - 338. [Abstract] [Full Text] [PDF]

				E. Rodriguez-Miranda, M. G Buffone, S. E Edwards, T. S Ord, K. Lin, M. D Sammel, G. L Gerton, S. B Moss, and C. J Williams Extracellular Adenosine 5'-Triphosphate Alters Motility and Improves the Fertilizing Capability of Mouse Sperm Biol Reprod, July 1, 2008; 79(1): 164 - 171. [Abstract] [Full Text] [PDF]

				A. R. Brennan, B. Dolinsky, M.-A. T. Vu, M. Stanley, M. F. Yeckel, and A. F.T. Arnsten Blockade of IP3-mediated SK channel signaling in the rat medial prefrontal cortex improves spatial working memory Learn. Mem., February 19, 2008; 15(3): 93 - 96. [Abstract] [Full Text] [PDF]

				T. G. Paunescu, N. Da Silva, L. M. Russo, M. McKee, H. A. J. Lu, S. Breton, and D. Brown Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells Am J Physiol Renal Physiol, January 1, 2008; 294(1): F130 - F138. [Abstract] [Full Text] [PDF]

				S. E. Edwards, M. G. Buffone, G. R. Knee, M. Rossato, G. Bonanni, S. Masiero, S. Ferasin, G. L. Gerton, S. B. Moss, and C. J. Williams Effects of Extracellular Adenosine 5'-Triphosphate on Human Sperm Motility Reproductive Sciences, October 1, 2007; 14(7): 655 - 666. [Abstract] [PDF]

				K. Schlingmann, M. A. Michaut, J. L. Mcelwee, C. A. Wolff, A. J. Travis, and R. M. Turner Calmodulin and CaMKII in the Sperm Principal Piece: Evidence for a Motility-Related Calcium/Calmodulin Pathway J Androl, September 1, 2007; 28(5): 706 - 716. [Abstract] [Full Text] [PDF]

				A. Schmid, Z. Sutto, M.-C. Nlend, G. Horvath, N. Schmid, J. Buck, L. R. Levin, G. E. Conner, N. Fregien, and M. Salathe Soluble Adenylyl Cyclase Is Localized to Cilia and Contributes to Ciliary Beat Frequency Regulation via Production of cAMP J. Gen. Physiol., July 1, 2007; 130(1): 99 - 109. [Abstract] [Full Text] [PDF]

				D. Wang, J. Hu, I. A. Bobulescu, T. A. Quill, P. McLeroy, O. W. Moe, and D. L. Garbers A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC) PNAS, May 29, 2007; 104(22): 9325 - 9330. [Abstract] [Full Text] [PDF]

				M. Kujala, S. Hihnala, J. Tienari, K. Kaunisto, J. Hastbacka, C. Holmberg, J. Kere, and P. Hoglund Expression of ion transport-associated proteins in human efferent and epididymal ducts Reproduction, April 1, 2007; 133(4): 775 - 784. [Abstract] [Full Text] [PDF]

				A. M. Stessin, J. H. Zippin, M. Kamenetsky, K. C. Hess, J. Buck, and L. R. Levin Soluble Adenylyl Cyclase Mediates Nerve Growth Factor-induced Activation of Rap1 J. Biol. Chem., June 23, 2006; 281(25): 17253 - 17258. [Abstract] [Full Text] [PDF]

				A. Darszon, J. J Acevedo, B. E Galindo, E. O Hernandez-Gonzalez, T. Nishigaki, C. L Trevino, C. Wood, and C. Beltran Sperm channel diversity and functional multiplicity. Reproduction, June 1, 2006; 131(6): 977 - 988. [Abstract] [Full Text] [PDF]

				L. R. Fraser, E. Beyret, S. R. Milligan, and S. A. Adeoya-Osiguwa Effects of estrogenic xenobiotics on human and mouse spermatozoa Hum. Reprod., May 1, 2006; 21(5): 1184 - 1193. [Abstract] [Full Text] [PDF]

				M. T. Branham, L. S. Mayorga, and C. N. Tomes Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway J. Biol. Chem., March 31, 2006; 281(13): 8656 - 8666. [Abstract] [Full Text] [PDF]

				C. Steegborn, T. N. Litvin, K. C. Hess, A. B. Capper, R. Taussig, J. Buck, L. R. Levin, and H. Wu A Novel Mechanism for Adenylyl Cyclase Inhibition from the Crystal Structure of Its Complex with Catechol Estrogen J. Biol. Chem., September 9, 2005; 280(36): 31754 - 31759. [Abstract] [Full Text] [PDF]

				H. Han, A. Stessin, J. Roberts, K. Hess, N. Gautam, M. Kamenetsky, O. Lou, E. Hyde, N. Nathan, W. A. Muller, et al. Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils J. Exp. Med., August 1, 2005; 202(3): 353 - 361. [Abstract] [Full Text] [PDF]

				S. Masuda and T.-a. Ono Adenylyl Cyclase Activity of Cya1 from the Cyanobacterium Synechocystis sp. Strain PCC 6803 Is Inhibited by Bicarbonate J. Bacteriol., July 15, 2005; 187(14): 5032 - 5035. [Abstract] [Full Text] [PDF]

				W. Geng, Z. Wang, J. Zhang, B. Y. Reed, C. Y. C. Pak, and O. W. Moe Cloning and characterization of the human soluble adenylyl cyclase Am J Physiol Cell Physiol, June 1, 2005; 288(6): C1305 - C1316. [Abstract] [Full Text] [PDF]

				V. Beaulieu, N. Da Silva, N. Pastor-Soler, C. R. Brown, P. J. S. Smith, D. Brown, and S. Breton Modulation of the Actin Cytoskeleton via Gelsolin Regulates Vacuolar H+-ATPase Recycling J. Biol. Chem., March 4, 2005; 280(9): 8452 - 8463. [Abstract] [Full Text] [PDF]

				M. Luconi, I. Porazzi, P. Ferruzzi, S. Marchiani, G. Forti, and E. Baldi Tyrosine Phosphorylation of the A Kinase Anchoring Protein 3 (AKAP3) and Soluble Adenylate Cyclase Are Involved in the Increase of Human Sperm Motility by Bicarbonate Biol Reprod, January 1, 2005; 72(1): 22 - 32. [Abstract] [Full Text] [PDF]

				Z. Sutto, G. E Conner, and M. Salathe Regulation of human airway ciliary beat frequency by intracellular pH J. Physiol., October 15, 2004; 560(2): 519 - 532. [Abstract] [Full Text] [PDF]

				B. Marquez and S. S. Suarez Different Signaling Pathways in Bovine Sperm Regulate Capacitationand Hyperactivation Biol Reprod, June 1, 2004; 70(6): 1626 - 1633. [Abstract] [Full Text] [PDF]