Adenylyl Cyclase, a Coincidence Detector for Nitric Oxide

Abstract

Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the V max of the enzyme without altering the K m for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and anS-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase. Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the V max of the enzyme without altering the K m for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and anS-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase. nitric oxide calmodulin guanine nucleotide-binding protein that stimulates adenylyl cyclase guanine nucleotide-binding protein that inhibits adenylyl cyclase phosphodiesterase nitric oxide synthetase S-nitroso-N-acetyl-d,l-penicillamine sodium nitroprusside prostaglandin E1 glyceraldehyde-3-phosphate dehydrogenase dithiothreitol adenosine 5′-(β,γ-imino)triphosphate Nitric oxide (NO)1 has been attributed roles in a variety of cellular activities throughout the body. For example, NO has been implicated as a regulator of vasodilation, synaptic plasticity, and immune defense. In each case, however, NO has potentially deleterious effects should its production be disturbed, as occurs in excitotoxicity and ischemia (reviewed in Refs. 1Garthwaite J. Trends Neurosci. 1991; 14: 60-67Abstract Full Text PDF PubMed Scopus (2337) Google Scholar, 2Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2147) Google Scholar, 3Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1635) Google Scholar). To understand the means by which NO achieves its paradoxical effects, and eventually to control its actions, attempts have focused on characterizing the enzymes responsible for NO production (NOS), identifying target molecules that are altered by NO, and specifying the underlying mechanism(s) by which NO alters those targets. One target of NO is the soluble guanylyl cyclase. It is the subsequent increase in cGMP levels that is believed to mediate NO-induced vasodilation. Given that an activation of soluble guanylyl cyclase occurs almost universally in response to NO, changes in cGMP have received primary consideration as the mechanism by which NO acts. However, the multiplicity of NO actions are unlikely to be explained by a common mechanism and a number of laboratories have suggested alternative targets of NO. Generally, cellular components have been implicated as targets of NO based largely on the ability of NO or NO-releasing compounds to alter their activities in vitro. Thus, whether they are altered in intact cells in response to NO, or what the possible physiological consequences may be, remain speculative. Our approach to discerning how NO functions has been to examine its effects on intact cells. We previously demonstrated that NO inhibits the production of cAMP pulses in Dictyostelium discoideum and does so independently of any changes in guanylyl cyclase activity (4Tao Y.-P. Howlett A. Klein C. FEBS Lett. 1992; 314: 49-52Crossref PubMed Scopus (21) Google Scholar, 5Tao Y.-P. Howlett A. Klein C. Cell Signal. 1996; 8: 27-34Crossref Scopus (9) Google Scholar). The sum of the data indicated that NO specifically alters either a regulatory domain of the adenylyl cyclase itself or a distinct regulatory moiety. We have also observed that the addition of NO gas or NO donor compounds to cultures of N18TG2 neuroblastoma cells inhibits Gs-coupled and forskolin-stimulated cAMP accumulation (6Tao Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar). NO-mediated inhibition of forskolin-stimulated cAMP production is unaltered by pretreating cells with pertussis toxin, implying that the inhibition did not involve Gi (6Tao Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar). Although experiments using intact cells were able to eliminate a number of potential targets that NO could modulate that would inhibit cAMP production, a number of possible targets still remained. In particular, NO has been proposed to inhibit GAPDH and in so doing limit the cellular levels of ATP (7Dimmeler S. Brune B. Eur. J. Biochem. 1992; 210: 305-310Crossref PubMed Scopus (57) Google Scholar). Similarly, NO-stimulated poly-(ADP-ribosylation) has been proposed to limit ATP production as a result of decreased availability of NAD (8Zhang J. Dawson V.L. Dawson T.M. Snyder S. Science. 1994; 263: 687-689Crossref PubMed Scopus (1085) Google Scholar). If such events were to occur in N18TG2 cells in response to NO, the cell's ability to produce cAMP could be compromised. NO has also been shown to alter the activity of ion channels (9Zufall F. Shepherd G.M. Barnstable C.J. Curr. Opin. Neurobiol. 1997; 7: 404-412Crossref PubMed Scopus (99) Google Scholar). Because a number of adenylyl cyclase isoforms are regulated by Ca2+ (10Mons N. Cooper D.M.F. Trends Neurosci. 1995; 18: 536-542Abstract Full Text PDF PubMed Scopus (117) Google Scholar), this could potentially influence the activity of the activity in N18TG2 cells. For example, the type V and VI isoforms are inhibited by Ca2+ by what appears to be the binding of the cation directly to the enzyme (11Scholich K. Barbier A.J. Mullenix J.B. Patel T.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2915-2920Crossref PubMed Scopus (64) Google Scholar). Thus, to further address the mechanism by which NO regulates cAMP production in N18TG2 cells, and to identify the target of its actions, we have studied the effects of NO on adenylyl cyclase activity in purified plasma membrane preparations. In the present report, we identify the predominant isoform of adenylyl cyclase present in N18TG2 cells and characterize its inhibition by NO in purified plasma membranes. Our findings demonstrate a novel, isoform-specific regulation of adenylyl cyclase activity by NO. Sucrose gradient purified plasma membranes (12Howlett A.C. Mol. Pharmacol. 1985; 27: 429-436PubMed Google Scholar) were resuspended in buffer A (50 mm NaHepes, pH 8, 3 mm MgCl2, 1 mm EDTA) at 0.5 mg/ml. Protein determinations were done according to Bradford (13Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Unless indicated otherwise, membranes were preincubated with 3 mmSNP for 30 min on ice. Other reagents were present as described in the individual experiments. Where indicated, membranes were washed free of any additions by diluting them 10-fold in ice-cold buffer A, centrifuging at 700 × g for 10 min, and resuspending them in ice-cold buffer A at 0.5 mg/ml. Adenylyl cyclase activity was assayed by incubating membranes at 30 °C for 20 min and monitoring the conversion of [α-32P]ATP to [32P]cAMP at 30 °C (12Howlett A.C. Mol. Pharmacol. 1985; 27: 429-436PubMed Google Scholar, 14Bidaut-Russell M. Howlett A.C. Neurochem. Int. 1987; 11: 287-292Crossref PubMed Scopus (4) Google Scholar, 15Howlett A.C. Qualy J.M. Khachatrian L.L. Mol. Pharmacol. 1986; 29: 307-313PubMed Google Scholar). The standard reaction mixture contained 50 mm NaHepes, pH 8, 1 mm EDTA, 10 mmMgCl2, 0.5 mm ATP containing 106cpm of [α-32P]ATP, 0.1 mm cAMP containing 10,000 cpm of [3H]cAMP, 0.1 mm Ro20-1724, 1.5 mm K+ phosphoenolpyruvate, 10 μg/ml pyruvate kinase, 0.1 mg/ml bovine serum albumin, and 25 μg of membrane protein in a final volume of 100 μl. Triplicate samples are assayed. Enzyme activity is proportional to the concentration of membranes and the time of incubation (12Howlett A.C. Mol. Pharmacol. 1985; 27: 429-436PubMed Google Scholar). Nuclei were removed and total cytoplasmic RNA was isolated according to Maniatis et al.(16Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: Scholar). RNA was on and to membranes. were with using the were for adenylyl cyclase isoforms and The a that was to was also by of the were done in Anal. Biochem. 1986; PubMed Scopus Google Scholar) at were washed in × and in 1 × at °C and to or for 1 cyclase 1 × is m m pH nitroprusside were from and were from and was from and was from reagents were from membranes were preincubated with of which they were assayed for forskolin-stimulated adenylyl cyclase activity was also in the of and be in a in of membranes for 30 min with the response of adenylyl cyclase to and did so in a inhibition was observed membranes were preincubated with 0.1 mm inhibition was present at 3 The inhibition 50 and with membrane a of inhibition to that with intact cells (6Tao Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar). enzyme activity is indicated for membranes that were not and for membranes with 10 mm Although in experiments we did an inhibition of enzyme in this inhibition was for in To that the inhibition of forskolin-stimulated adenylyl cyclase activity the of NO and not of we preincubated membranes with a distinct NO also inhibited adenylyl cyclase by in a In NO donors were in their ability to so not Other experiments indicated that the effects of NO donors were unaltered membranes were preincubated in the of or not It would that NO, an is responsible for the inhibition of adenylyl cyclase The that the effects of NO in intact cells (6Tao Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar) be in purified plasma membrane preparations the possible targets of NO to components in those preparations. using intact cells indicated that changes in cGMP or did not mediate the inhibition of cAMP accumulation in response to or (6Tao Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar). Although we not soluble guanylyl cyclase to be present in membrane we the of of cGMP by cGMP to the adenylyl cyclase reaction changes in enzyme activity were observed not of for the in adenylyl cyclase activity in response to NO in purified membrane preparations are in the a for the K m isoform that is present in N18TG2 cells A.C. Mol. Pharmacol. Google Scholar), and an standard is to for any of To identify the target of actions on adenylyl cyclase activity in plasma we a of the effects of NO on the regulation of the enzyme by and In the shown in the ability of to hormone- and forskolin-stimulated adenylyl cyclase activity was at of did not alter the for or Thus, the binding of to its or of to the adenylyl was unaltered by NO. In of were able to enzyme stimulates adenylyl cyclase by directly binding to the and the protein Although does not has been shown that activation to of the enzyme 1994; PubMed Scopus Google Scholar). The addition of to the adenylyl cyclase activity that with We observed a increase in enzyme was whether or not membranes been with It would from this that is a of proteins in membrane preparations that be by the addition of to adenylyl cyclase activity and that does not alter this have been shown to the activation of by the in the of a L. J. Full Text PDF PubMed Google Scholar, Howlett A.C. Neurosci. 1987; Scopus Google Scholar). with previously data L. J. Full Text PDF PubMed Google Scholar), 3 that the of adenylyl cyclase activity was with of MgCl2, in the of 3 to shown is that this enzyme was not altered in membranes that been with enzyme activity was These data would that the cation regulatory was not by NO. In the indicate that the ability of to adenylyl cyclase is not altered by with NO could adenylyl cyclase activity by the enzyme directly or by altering the of a distinct regulatory To identify possible regulatory proteins of the enzyme present in N18TG2 cells and to their in this we the isoform present in N18TG2 cells. The that this would be a type V or VI isoform was suggested by its The of a regulation of cAMP production in N18TG2 cells (6Tao Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar) was plasma membranes were with and in adenylyl cyclase or in the ability of NO to inhibit that was would indicate that the enzyme is not of the type or isoform (10Mons N. Cooper D.M.F. Trends Neurosci. 1995; 18: 536-542Abstract Full Text PDF PubMed Scopus (117) Google Scholar, J. 7: PubMed Scopus Google Scholar, Annu. Rev. Pharmacol. 1996; PubMed Scopus Google Scholar). The enzyme in N18TG2 cells is regulated by that and inhibit Gi (12Howlett A.C. Mol. Pharmacol. 1985; 27: 429-436PubMed Google Scholar, 14Bidaut-Russell M. Howlett A.C. Neurochem. Int. 1987; 11: 287-292Crossref PubMed Scopus (4) Google Scholar, 15Howlett A.C. Qualy J.M. Khachatrian L.L. Mol. Pharmacol. 1986; 29: 307-313PubMed Google Scholar, M. Proc. Natl. Acad. Sci. U. S. A. 72: PubMed Scopus Google Scholar), a of the type VI isoform (10Mons N. Cooper D.M.F. Trends Neurosci. 1995; 18: 536-542Abstract Full Text PDF PubMed Scopus (117) Google Scholar, J. 7: PubMed Scopus Google Scholar, Annu. Rev. Pharmacol. 1996; PubMed Scopus Google Scholar). with the of the and for which of cAMP production in response to (10Mons N. Cooper D.M.F. Trends Neurosci. 1995; 18: 536-542Abstract Full Text PDF PubMed Scopus (117) Google J. 7: PubMed Scopus Google Scholar, Annu. Rev. Pharmacol. 1996; PubMed Scopus Google Scholar). the adenylyl cyclase in N18TG2 cells is by which from the type isoform Annu. Rev. Pharmacol. 1996; PubMed Scopus Google Scholar). To the data and to which of the type V and VI adenylyl cyclase is predominant in N18TG2 cells, we Northern of N18TG2 RNA with the for the type V and VI in a was observed the blot was with the type VI not the type V was to the type and isoforms were 1 If an isoform the type VI is in cells, is present at a that is or by Northern blot analyses and would not influence the of data the regulation by NO. We preincubated membranes with and forskolin-stimulated adenylyl cyclase activity at of The are in The of the of indicated that m did not control and The were and mm for control and would that the binding of was not by the of NO. However, max for membranes was to that in control membranes of a of the enzyme conversion of to Although inhibited adenylyl cyclase membranes were preincubated with the NO inhibition was observed was at the of the that the to which inhibits enzyme activity was the of the In that membranes were preincubated with for the indicated and assayed for of inhibition could be a inhibition by One of is that does not inhibit the adenylyl cyclase by altering a in the was in experiments in which membranes were washed free of the to the The of inhibition of forskolin-stimulated adenylyl cyclase activity was unaltered with membranes 8, of reducing to the effects of were preincubated with 3 mmSNP for 30 were washed free of and for 20 min in the or of the indicated of or adenylyl cyclase activity was The are to the forskolin-stimulated adenylyl cyclase activity in membranes that not been preincubated with or reducing The data are of of or 1 to adenylyl cyclase from were preincubated with or without Where indicated, either or was also membranes were preincubated with was also The or of did not alter the 30 min membranes were washed and assayed for or adenylyl cyclase with present in the activity was and in control and membranes. The data are of of adenylyl cyclase activity by of the substrate-binding were preincubated with or without 3 mm in the or of the indicated the membranes were washed and resuspended in buffer as described were assayed for forskolin-stimulated adenylyl cyclase was to the membranes were preincubated in the of any additions activities were and in control and The data are of experiments. and indicated that the for and membranes were not that they were from the for and and that the for the were not from each The that was at the time of the also the that a of the reaction mixture to modulate the effects of NO. and VI adenylyl the for protein A, and incubation of those isoforms with protein A alters their protein has also been shown to adenylyl altering their activities T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: Scholar, J. 7: PubMed Scopus Google Scholar, Annu. Rev. Pharmacol. 1996; PubMed Scopus Google Scholar). To the that a mechanism alter the response to NO, which as a ATP as the for adenylyl cyclase. or ATP as inhibition was observed not that a does not modulate the ability of NO to inhibit adenylyl cyclase. We the that of the substrate-binding site of the enzyme the effects of NO. To we the of ATP the could limit the ability of to inhibit the In the shown in membranes were preincubated with in the or of the indicated were washed and assayed for forskolin-stimulated In the of any in a of enzyme Adenylyl cyclase activity was the effects of was present the The was for ATP were In was also to the enzyme was present at the time of that the effects of ATP were not to a It would that the conformation of the enzyme of the substrate-binding site of the enzyme to the effects of NO. Given we the conformation of the as by either or could also the enzyme the effects of NO. To membranes were preincubated with in the or of or membranes were preincubated with was also to activation of the membranes were washed and assayed for their or shown in of control membranes with either or did not alter their effects on adenylyl cyclase activity to the enzyme Similarly, the inhibition of and activity was unaltered were present the in the of ATP. We previously in intact N18TG2 cells, that NO-mediated inhibition of cAMP accumulation in response to or is ability to to for 20 min in the of NO (6Tao Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar). shown in membranes were washed free of and for 20 min in forskolin-stimulated adenylyl cyclase activity However, washed membranes were with activity was in a effects were observed Cys was of In did not for of forskolin-stimulated activity was itself inhibitory to the We have that 10 mm Cys alter adenylyl cyclase activity directly to the Thus, the NO-mediated inhibition of adenylyl cyclase activity in plasma membranes is an reducing is We also observed that the ability of to the effects of NO are membranes were for with 10 mm to the of forskolin-stimulated adenylyl cyclase was observed at min, the time not isoforms of the enzyme have been each with distinct regulatory as in of the enzyme in or in experiments (reviewed in Refs. N. Cooper D.M.F. Trends Neurosci. 1995; 18: 536-542Abstract Full Text PDF PubMed Scopus (117) Google Scholar, J. 7: PubMed Scopus Google Scholar, and Annu. Rev. Pharmacol. 1996; PubMed Scopus Google Scholar). the that each enzyme is regulated by a variety of the of a mechanism of adenylyl cyclase regulation has been by the that changes in adenylyl cyclase activity an of the of isoforms present in a In the present we have the predominant isoform present in N18TG2 cells as a type VI enzyme and have described its novel regulation by NO. We previously demonstrated that of intact N18TG2 cells with NO or NO donors their production of cAMP with or (6Tao Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar). The effects of NO be in purified plasma membrane its possible targets to components in those preparations. Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar, and this guanylyl and protein as NO targets. In so we have the known of the type VI isoform as targets of NO. we are able to demonstrate the of adenylyl cyclase activity in plasma membranes also events such as changes in ATP levels or ion as possible of the effects of NO on the We that NO functions either an as novel regulator of the adenylyl cyclase in N18TG2 cells or that the enzyme itself is the target of NO. It is of to that isoform of adenylyl cyclase may also be regulated by NO. et J. 1994; Full Text PDF PubMed Google Scholar) that the regulation of type adenylyl cyclase in membranes from cells is inhibited by NO enzyme activity is The proposed that NO may by a Cys residue(s) at the site of the adenylyl cyclase. et T. L. F. S. T. PubMed Scopus Google Scholar) a of the type adenylyl cyclase that binding a that Cys is or of is by NO, or NO functions to inhibit activation by of the experiments were to analyses of also to be NO regulates type adenylyl cyclase in either membranes or in intact cells that the enzyme this in a in its activation by with the effects that we have observed in which NO inhibits the forskolin-stimulated cAMP in intact N18TG2 cells and in purified plasma and does so in a that is of any regulation of the of soluble of adenylyl cyclase have indicated that the enzyme in by the binding of such as or by of the site Science. 1997; PubMed Scopus Google Scholar, Mol. Pharmacol. 1998; PubMed Scopus Google Scholar). Our analyses indicated that the inhibition by NO an suggesting that the conformation of the enzyme is altered in a that its The inhibitory effects of NO were not altered membranes were preincubated with either or Thus, the of the enzyme that are as a result of or binding not alter the ability of NO to inhibit adenylyl cyclase. Occupancy of the however, did the enzyme from the inhibition by NO. would those the by NO is either is ATP binding or the enzyme conformation ATP binding would the to be the If the site contained the by NO, we would that cyclase activity would also be inhibited by NO. was not the Given the the of the we would also isoforms to be regulated by NO. We that the activity of the type enzyme is not altered by NO J. 1994; Full Text PDF PubMed Google Scholar), suggesting in their the type and VI isoforms a conformation that to NO. In to the type VI would that the type isoform of adenylyl in which that modulate binding are to NO, in a conformation such that NO able to inhibit The that of the substrate-binding site of the enzyme protects from by NO would indicate that of cAMP production occur to NO of the enzyme by a adenylyl cyclase or is with that of NO, the inhibitory effects of NO be Our data indicate that the effects of NO be also by the ability of cells to the inhibition of adenylyl cyclase. intact cells, the inhibitory effects of NO on cAMP accumulation are reversed cells are in the of NO donors (6Tao Y.-P. Najafi L. Shipley S. Howlett A. Klein C. J. Pharmacol. Exp. Ther. 1998; 286: 298-304PubMed Google Scholar). In plasma membrane the of NO donor compounds did not the inhibition of adenylyl cyclase suggesting that a The NO-mediated however, was reversed membranes were washed free of NO donors and in the of a reducing either or a physiological as the underlying mechanism of adenylyl cyclase inhibition J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1635) Google Scholar). also indicate that the ability of NO to a cell's production of cAMP the cell's a of has to indicate that the cell's potential alter a variety of in the control of and N. B. C. Cold Spring Harbor 1996; Scholar). These data such to Given the of the effects of NO, is that the physiological consequences of NO with to cAMP production, the levels of NO and its If the of NO from the activity of a such levels be and The changes in a cell's to would also be and of a or levels of NO as by would result in a to In levels of NO are believed to be to the from and a number of (reviewed in J. Trends Neurosci. 1991; 14: 60-67Abstract Full Text PDF PubMed Scopus (2337) Google Scholar, 2Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2147) Google Scholar, 3Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1635) Google Scholar). In of their and of adenylyl have been proposed to as Cell. Full Text PDF Scopus Google Scholar), the enzymes are by and a in The response to could be type adenylyl cyclase to in a that could be by the of by Gi the response to may be by the of a the type V and VI isoforms are by inhibited by Ca2+ (10Mons N. Cooper D.M.F. Trends Neurosci. 1995; 18: 536-542Abstract Full Text PDF PubMed Scopus (117) Google Scholar). Our data indicate that the type VI isoform of adenylyl cyclase predominant in N18TG2 cells is a for NO and that the potential of the and the of influence the ability of NO to cAMP We T. for throughout the of and for the adenylyl cyclase for the for and for the