GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity , intracellular calcium , NOS , and guanylyl cyclase Guanosine 5 ' - triphosphate -LRB- GTP -RRB- is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal . Previous studies have proposed that GTP avoidance is linked to a receptor-mediated , calcium-based depolarization . However , the intracellular mechanisms involved in GTP avoidance have not been previously documented . In this study , we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila . Using behavioral assays , enzyme immunosorbent assays , Western blotting , and immunofluorescence , we present data that implicate a tyrosine kinase , phospholipase C , intracellular calcium , nitric oxide synthase -LRB- NOS -RRB- and guanylyl cyclase in GTP signaling . The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays . Similarly , pharmacological inhibitors of phospholipase C , NOS , and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP . Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia , suggesting that this enzyme activity could be directly involved in ciliary reversal . Introduction Chemorepellents are molecules that inhibit or repel the migration of individual cells . In protozoans , chemorepellents are hypothesized to serve a defense mechanism , helping the organism to avoid predation and environmental hazards . For example , a damaged Tetrahymena thermophila cell may leak cytosolic purines such as adenosine triphosphate -LRB- ATP -RRB- or guanosine 5 ' - triphosphate -LRB- GTP -RRB- into the surrounding environment , causing other cells to avoid the area , thereby avoiding the source of damage to the original cell and increasing the survival rate of the species -LSB- 1 , 2 -RSB- . It is possible that cytosolic purines may be secreted by these organisms as a means of communication between cells -LRB- analogous to paracrine secretion -RRB- or in order to trigger second-messenger pathways within the cell doing the secretion -LRB- analogous to autocrine secretion -RRB- . At this time , secretion of purine nucleotide by Tetrahymena has not been documented , although Tetrahymena have been shown to secrete a number of enzymes and peptides . In T. thermophila , extracellular GTP is a known chemorepellent -LSB- 1 , 3 , 4 -RSB- . GTP causes depolarization in the somatic membrane -LSB- 2 -RSB- , which has been proposed to trigger opening of the voltage-dependent calcium channels in the ciliary membrane -LSB- 2 -RSB- . The increase in Ca2 + concentration causes the cilia to reverse their direction -LSB- 1 -RSB- , resulting in a disruption of normal swimming . This `` avoidance reaction '' -LSB- 1 -RSB- is marked by jerky movements , backward swimming , or spinning . This chemokinetic response involves a series of avoidance reactions , resulting in a `` biased random walk , '' which orients the organism away from the source of the repellent -LSB- 3 -RSB- . Avoidance to chemorepellents may be quantified by observing the response of single cells exposed to repellent using a dissection microscope . T. thermophila avoid micromolar concentrations of GTP -LSB- 1 -RSB- . An in vivo binding assay using -LSB- 32P -RSB- GTP showed the KD for GTP binding to intact T. thermophila is 21 ± 2.5 nM . GTP binds to its receptor with high affinity , and binding is saturable as well as reversible -LSB- 1 -RSB- , all of which are characteristic of membrane receptors . Fluorescent staining with 2 ′ - -LRB- or 3 ′ -RRB- - O - -LRB- 2,4,6-trinitrophenyl -RRB- -LRB- TNP -RRB- - GTP has shown GTP binding on the cilia and cell surface of T. thermophila -LSB- 4 -RSB- . However , the second messenger pathways involved in GTP signaling have not been previously described . Recently , Iwamoto and Nakaoka -LSB- 4 -RSB- found that addition of extracellular GTP to the medium induces cell division in T. thermophila , a result consistent with a tyrosine kinase signaling mechanism , as tyrosine kinases are involved in many major mitotic pathways . Tyrosine phosphorylation has previously been shown to modulate the interaction of calmodulin with target proteins , including protein phosphatase 2B -LRB- calcineurin -RRB- and neuronal nitric oxide synthase -LRB- nNOS -RRB- in an in vitro assay -LSB- 5 -RSB- . Tyrosine kinase activity has also been shown to activate phospholipase C , NOS -LRB- via protein kinase C -RRB- , and guanylyl cyclase in the rat ileum -LSB- 6 -RSB- . Tyrosine kinases are frequently associated with activation of phospholipase C-γ , and with release of calcium from internal stores via the IP3 pathway -LSB- 7 -RSB- . In our current study , we examined the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila . Our data confirm this hypothesis . In addition to tyrosine kinase signaling , we found that phospholipase C , intracellular calcium stores , NOS , and guanylyl cyclase are necessary for behavioral avoidance to occur . All of these second messengers are consistent with the existence of a tyrosine kinase signaling pathway in T. thermophila . Materials and methods Cell cultures T. thermophila , strain B2086 , a generous gift from T.M. Hennessey -LRB- SUNY-Buffalo -RRB- were used for all of the experiments . Cells were grown at 25 °C in the axenic medium of Dentler -LSB- 8 -RSB- without shaking . Two-day-old cell cultures were used for all assays described below . Chemicals and solutions Behavioral assays were carried out in a buffer of pH 7.0 containing 10 mM Trizma base , 0.5 mM MOPS , and 50 μM CaCl2 . All repellents and inhibitors used were dissolved in this buffer . Compounds that were insoluble in aqueous solutions were first dissolved in a small quantity of dimethylsulfoxide -LRB- DMSO -RRB- and then diluted 1:1,000 or more into the buffer and vortexed vigorously . The chemorepellent guanosine-5 ′ - O - -LRB- 3-thio -RRB- triphosphate -LRB- GTP-γ-S -RRB- was obtained from Alexis Biochemicals -LRB- San Diego , CA , USA -RRB- , along with the G-protein inhibitor pertussis toxin . The tyrosine kinase inhibitor genistein was obtained from Acros Organics -LRB- New Jersey , USA -RRB- . Calphostin C -LSB- a protein kinase C -LRB- PKC -RRB- inhibitor -RSB- , diadzein a control for genistein inhibition -RRB- , guanosine-5 ′ - O - -LRB- 2-thiodiphosphate -RRB- -LRB- GDP-β-S -RRB- -LRB- a G-protein inhibitor -RRB- , Rp-8-Bromoguanosine-3 ′ ,5 ′ - cyclic monophosphorothioate -LRB- Rp-8-Br-cGMPs -RRB- -LRB- a competitive inhibitor of PKG -RRB- , 1 - -LRB- 6 - -LRB- 17β-3-methoxyestra-1,3,5 -LRB- 10 -RRB- - trien-17-yl -RRB- amino -RRB- hexyl -RRB- -1 H-pyrrole-2,5-dione -LRB- U73122 -RRB- -LRB- a phospholipase C inhibitor -RRB- , NG-monomethyl-L-arginine -LRB- L-NMMA -RRB- , and N - -LRB- 3 - -LRB- Aminomethyl -RRB- benzyl -RRB- acetamidine -LRB- 1400W -RRB- -LRB- NOS inhibitors -RRB- , 4H-8-bromo-1,2,4-oxadiazolo -LRB- 3,4-d -RRB- benz -LRB- b -RRB- -LRB- 1,4 -RRB- oxazin-1-one -LRB- NS2028 -RRB- -LRB- a PKG inhibitor -RRB- , and the Quantizyme nitric oxide -LRB- NO -RRB- assay kit were purchased from BIOMOL Research Laboratories -LRB- Plymouth Meeting , PA , USA -RRB- . The polyclonal antiphosphotyrosine antibody was purchased from Upstate -LRB- Lake Placid , NY , USA -RRB- ; 30X Tris-buffered saline -LRB- TBS -RRB- and 30X TBS/nonidet P40 -LRB- NP40 -RRB- were obtained from Modern Biology -LRB- West Lafayette , IN , USA -RRB- . The guanosine 3 ′ 3 ; 5 ′ monophosphate -LRB- cGMP -RRB- emzyme immunosorbent assay -LRB- EIA -RRB- kit was purchased from Cayman Chemical -LRB- Ann Arbor , MI , USA -RRB- . Sodium metavanadate -LRB- a phosphatase inhibitor -RRB- and theophylline -LRB- a phosphodiesterase inhibitor -RRB- were obtained from Sigma Chemical -LRB- St. Louis , MO , USA -RRB- . All other chemicals were purchased from Sigma as well . Behavioral assays In vivo behavioral assays were carried out as previously described -LSB- 1 , 9 , 10 -RSB- . Briefly , cells were washed in buffer , and 300 μl of cell suspension was transferred to the first well of a three-well spot microtiter plate . Cells were then individually transferred by micropipette to the second well , which contained 300 μl of the test concentration of inhibitor dissolved in buffer . After at least 10 -- 15 min of incubation time , individual cells were transferred to the third well , which contained 300 μl GTP-γ-S and the test concentration of inhibitor . Each cell was briefly observed -LRB- 1 -- 5 s -RRB- for signs of avoidance . Avoidance behavior was characterized by jerky , backward movements ; swimming in small , tight circles ; sudden reversal of swimming direction ; or any deviation from the normal helical swimming pattern of T. thermophila . Western blots Western blots were performed as follows : For each sample , 10 ml of the same 2-day-old culture was washed 3 × in behavioral buffer ; 100 μM GTP-γ-S -LRB- the concentration required to elicit 100 % avoidance -RRB- was added to the tube of cells marked GTP . The control tube received only buffer ; no GTP was added . Both tubes were immediately extracted with 0.1 % sodium dodecyl sulphate -LRB- SDS -RRB- in the presence of 1 mM sodium orthovanadate for 30 min at 4 °C . Extracts were spun at 16,000 g at 4 °C for 30 min to precipitate insoluble components . Supernatant was collected , and 5 × loading buffer was applied in preparation for SDS polyacrylamide gel electrophoresis -LRB- PAGE -RRB- ; 100 μl of each sample was applied to a 10 % SDS-PAGE . Amount of protein loaded onto the gel was standardized using the BIO-RAD protein assay ; in addition , a control , Coomassie-stained gel , was run to show that equal amounts of protein were loaded into each well . Proteins were transferred from the SDS-PAGE to nitrocellulose for 30 min at 150 mA . Blots were then blocked overnight in a 3 % solution of powdered milk . Blots were incubated with a 1:1,000 dilution of polyclonal phosphotyrosine antibody in the presence of 3 % powdered milk for 1.5 h , washed 3 × in TBS , then incubated with a 1:100 dilution of alkaline phosphate conjugated anti-rabbit antibody for 1.5 h in the presence of 3 % powdered milk . Blots were then washed 3 × with TBS and incubated for 5 min in an alkaline phosphatase buffer containing 100 mM NaCl , 5 mM MgCl2 , 100 mM Trizma base , pH 9.5 with hydrogen chloride -LRB- HCl -RRB- . Alkaline phosphatase buffer was then removed , and blots were developed using the Sigma-Fast system until bands were visible . EIA assays NO assays were carried out using 2-day-old cell cultures . Cells were washed twice in buffer and diluted to a final concentration of approximately 720 cells/ml . Cells were exposed to 100 μM GTP-γ-S for approximately 30 s , then spun down in a bench-top microcentrifuge for 30 s. Control cells were spun under identical conditions except that no GTP was added to the solution . Supernatant from both sets of cells was analyzed for NO using a kit from BIOMOL according to the manufacturer 's instructions . cGMP assays were carried out using 2-day-old cell cultures . Cells were concentrated to approximately 7.66 × 106 cells/ml in order to maximize cGMP production . Cells were exposed to 100 μM GTP for approximately 30 s , then immediately lysed by freezing in liquid nitrogen and thawing . Theophylline -LRB- 1 mM -RRB- was added to the lysate to inhibit phosphodiesterases . Lysate was spun at 16,000 g for 30 min . Control cells were treated in identical fashion except for the addition of GTP . Supernatant from both sets of cells was assayed for cGMP using a kit from Cayman Chemicals according to the manufacturer 's instructions . Immunofluorescence Ten milliliters of 2-day-old cells were starved overnight in behavioral buffer then washed 3 × in behavioral buffer and concentrated down into 2 ml . Mucocysts were removed by adding Alcian Blue GX to a final concentration of 0.05 % , then adding nine volumes of 0.25 % proteose peptone -LSB- 11 -RSB- . Mucocysts were allowed to precipitate by standing the tube in a rack for 10 min . Supernatant -LRB- including cells -RRB- was removed from the top of the tube . The cells were then washed 3 × in behavioral buffer to remove any remaining mucocysts or Alcian Blue and concentrated down into 3 ml total ; 1 ml of cells was set aside as the control cells . Another milliliter of cells was incubated with 100 μM GTP-γ-S . A third aliquot was incubated with 100 μg/ml genistein prior to GTP treatment . Immediately , all three aliquots of cells were fixed in 3.7 % formaldehyde -LRB- from 37 % stock diluted into behavioral buffer -RRB- with gentle shaking for 20 min . Cells were washed 3 × in 1 × TBS/NP40 -LRB- Modern Biology -RRB- then incubated with a 1:100 dilution of polyclonal antibody in 1 × TBS/NP40 for 1.5 h. Cells were again washed 3 × in 1 × TBS/NP40 then incubated in a 1:100 dilution of fluorescein isothiocyanate -LRB- FITC -RRB- - conjugated goat anti-rabbit IgG -LRB- ICN Biomedicals -RRB- for 1.5 h. Cells were washed 3 × in 1 × TBS before being viewed under a Nikon microscope at 400 × . Cells were photographed using a SPOT digital camera using a 10-s exposure for each photograph . Results GTP-γ-S is a chemorepellent in Tetrahymena , , with a minimum concentration of 100 μM required for 100 % avoidance -LRB- Fig. 1 -RRB- . This concentration was used in the inhibition studies that follow as well as in our biochemical studies . We used GTP-γ-S in our experiments rather than GTP to minimize the effects of the ecto-ATPase upon the assays -LSB- 3 -RSB- . However , the activity of apyrases and other ectoenzymes may still have degraded some of the GTP-γ-S before it could reach its intended receptor . The concentration of GTP-γ-S required in order to achieve 100 % avoidance in this study was 5 × higher than that previously published -LSB- 1 -RSB- ; however , considering that the cell line and growth conditions were different than those published earlier , this finding is not unexpected . The EC50 of GTP-γ-S in this study was approximately 30 μM . Fig. 1Guanosine-5 ′ - O - -LRB- 3-thio -RRB- triphosphate -LRB- GTP-γ-S -RRB- is an effective chemorepellent in Tetrahymena . In vivo behavioral bioassays -LRB- see `` Materials and methods '' -RRB- were used to show the concentration dependencies for avoidance reactions to GTP-γ-S . The percentage of cells showing avoidance was determined by observation of a single cell after transfer to the test solution . Each trial consisted of ten cells , which were individually scored as to whether or not avoidance occurred . Each point represents the mean ± standard deviation -LRB- SD -RRB- of at least three trials . Error bars , representing the standard deviation , are shown for each point . Minimum concentration required to give 100 % avoidance was 100 μM for GTP-γ-S . This concentration was used in the inhibition studies that follow . The EC50 of this compound was approximately 30 μM To determine whether a tyrosine kinase activity was involved in GTP avoidance , we used the tyrosine kinase inhibitor , genistein -LRB- Fig. 2 -RRB- . Genistein itself did not cause avoidance reactions , nor was it toxic to the cells . Baseline avoidance of ∼ 20 % -LSB- 9 -RSB- to 100 μM GTP-γ-S was seen at a genistein concentration of 100 μg/ml . The inhibition constant -LRB- IC -RRB- 50 of genistein was approximately 40 μg/ml . Another phytoestrogen , daidzein , served as a control for nonspecific side effects of genistein . As seen in Fig. 2 , this compound had no measurable effect on avoidance at the tested concentrations , suggesting that the inhibitory effect of genistein was indeed due to its tyrosine kinase inhibition and not attributable to some side effect of the drug . Fig. 2The tyrosine kinase inhibitor , genistein , blocks avoidance to 100 μM guanosine-5 ′ - O - -LRB- 3-thio -RRB- triphosphate -LRB- GTP-γ-S -RRB- in Tetrahymena . The phytoestrogens genistein -LRB- closed circles -RRB- and daidzein -LRB- open circles -RRB- were used as inhibitors of avoidance . Baseline avoidance was seen at a genistein concentration of 100 μg/ml . The IC50 of genistein was approximately 40 μg/ml . Daidzein served as a control for nonspecific effects of genistein and had no measurable effect on avoidance at the concentrations tested . Data shown represent the mean + / − standard deviation -LRB- SD -RRB- for three or more trials As an additional control for specificity , we tested whether genistein had any effect on the transduction of another chemorepellent , adenosine 5 ′ - O - -LRB- 3-thio -RRB- triphosphate -LRB- ATP-γ-S -RRB- . We used 150 μM ATP-γ-S , a concentration that normally causes 100 % avoidance in T. thermophila -LSB- 12 -RSB- . When cells exposed to ATP-γ-S were first incubated for 10 -- 15 min in 100 μg/ml genistein , no measurable effect on avoidance was seen . Cells continued to avoid 150 μM ATP-γ-S at a rate of 96.6 + 5.8 % -LRB- n ≥ 3 -RRB- . Genistein also failed to affect avoidance to the chemorepellent pituitary adenylate cyclase-activating polypeptide -LRB- PACAP -RRB- . When testing 0.1 μM PACAP , which normally causes 100 % avoidance in T. thermophila -LSB- 9 -RSB- , we found that prior incubation with 100 μg/ml genistein did not markedly affect avoidance . Cells continued to avoid 0.1 μM PACAP at a rate of 96.6 + 5.8 % -LRB- n ≥ 3 -RRB- . These data suggest that tyrosine kinase activation is specifically related to GTP exposure rather than being a general response to chemorepellent exposure . In an attempt to localize the phosphotyrosines within the cell , immunofluorescence experiments were performed using a polyclonal antiphosphotyrosine antibody . Three separate trials were done with this antibody , and representative photographs are compiled in Fig. 3a -- c. GTP-exposed cells -LRB- Fig. 3b -RRB- showed higher overall fluorescence intensity than did the controls and genistein-treated GTP-exposed cells -LRB- Fig. 3a , c -RRB- . Ciliary staining was seen in the GTP-exposed cells , along with punctuate staining of the cytosol . The control and genistein-treated GTP-exposed cells showed the same cytosolic staining pattern , but ciliary staining was not detected in these control groups , perhaps because the overall fluorescence level of these cells was so low . Fig. 3Guanosine 5 ' - triphosphate -LRB- GTP -RRB- exposure increases tyrosine kinase activity in Tetrahymena . a -- c Immunofluorescence labeling of phosphotyrosines in Tetrahymena is evidence of tyrosine kinase activity . Control -LRB- a -RRB- , GTP-exposed -LRB- b -RRB- , and genistein-treated GTP-exposed cells -LRB- c -RRB- were fixed and labeled with a polyclonal antiphosphotyrosine antibody . GTP-exposed cells showed higher fluorescence intensity than did control cells and cells treated with the tyrosine kinase inhibitor genistein . In addition , GTP-exposed cells showed ciliary staining in addition to punctuate staining of the cytosol . Total magnification 400 × . Cell length ∼ 50 μm . d Western blot of whole-cell extract obtained from control and GTP-exposed cells using a polyclonal antiphosphotyrosine antibody shows increased phosphorylation levels in extract taken from GTP-exposed cells -LRB- lane 2 -RRB- relative to extract from control cells -LRB- lane 3 -RRB- . A 66-kDa phosphoprotein stained similarly in both the GTP and control lanes , whereas bands of 42 , 35 , and 21 kDa were more heavily stained in the GTP lane relative to the control lane . Molecular weight markers are shown in the first lane -LRB- molecular weights in kDa -RRB- . A Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis -LRB- SDS-PAGE -RRB- run as a loading control showed equivalent staining of proteins in all lanes -LRB- not shown -RRB- To determine which proteins were being phosphorylated by the tyrosine kinase in response to GTP-γ-S , we also performed several Western blots of whole-cell extract obtained from control and GTP-γ-S - treated cells using the polyclonal antiphosphotyrosine antibody as a probe . Protein extracts were standardized so that lanes were loaded equally , and a control , Coomassie-stained gel , was also run to control for lane-loading artifacts -LRB- data not shown -RRB- . A representative Western blot is pictured in Fig. 3d . Both GTP-treated and control cells showed similar staining at 66 kDa . However , increased phosphotyrosine levels were seen in the GTP-γ-S-treated cells at 42 , 35 , and 21 kDa , consistent with tyrosine kinase activity . These bands were also present in the control cell extract ; however , the phosphorylation level in these lanes was so low that they were barely detectable with our staining procedure . Additional bands were also seen in both the GTP-exposed and the control lanes ; however , they were also too light to be measurable . A more sensitive assay , such as chemiluminescence based Western blot detection , might help to give a more complete profile of the phosphotyrosine-containing proteins shown in this procedure . In many cells , activation of a tyrosine kinase activates phospholipase C-γ . We used the phospholipase C inhibitor , U73122 , to determine whether phospholipase C might be involved in GTP avoidance . GTP avoidance was effectively eliminated at a U73122 concentration of 1 μM . This concentration was ten times lower than that used to inhibit phospholipase C in molluscan ciliary cells -LSB- 12 -RSB- . The IC50 of this compound was approximately 0.001 μM . Phospholipase C activates the PKC pathway through the generation of diacylglycerol -LRB- DAG -RRB- and inositol 1,4,5 trisphosphate -LRB- IP3 -RRB- . PKC is then activated by DAG binding in the presence of calcium . To determine whether PKC was involved in GTP avoidance , we used the PKC inhibitor calphostin C , which competes for DAG binding to PKC . In the presence of 10 μM calphostin C , avoidance to 100 μM GTP-γ-S was reduced only to 96.6 + / − 5.8 % , compared with 100 % avoidance in control cells . This concentration of calphostin C eliminated avoidance to 150 μM ATP-γ-S -LSB- 13 -RSB- . Because calphostin C had no measurable effect on GTP avoidance , we concluded that PKC activy is not required for GTP signaling . It is very likely that the two second messengers , IP3 and DAG , are generated by phospholipase C and that PKC is activated , but that PKC targets cellular substrates that are not directly involved in ciliary reversal . Because calcium signaling is often associated with tyrosine kinase pathways and because calcium influx is associated with GTP exposure -LSB- 2 -RSB- , we decided to eliminate calcium rom cells using the membrane-permeable calcium chelator 1,2-bis - -LRB- o-Aminophenoxy -RRB- - ethane-N , N , N ' , N ' - tetraacetic acid , tetra -LRB- acetoxymethyl -RRB- ester -LRB- BAPTA-AM -RRB- . As seen in Fig. 5a , exposure to this calcium chelator eliminated avoidance in a concentration-dependent manner , with baseline avoidance seen at a BAPTA-AM concentration of 5 μg/ml . This result was certainly not surprising , as all known chemorepellents that have been studied electrophysiologically in Tetrahymena have elicited depolarizations -LSB- 2 , 14 , 15 -RSB- that have been proposed to be linked to calcium . Because our data indicated that phospholipase C was involved in GTP avoidance , we decided to test the hypothesis that internal calcium stores may also contribute to avoidance , consistent with the liberation of IP3 from the plasma membrane . We used an inhibitor of the endoplasmic reticulum -LRB- ER -RRB- calcium ATPase , thapsigargin , to deplete ER calcium stores to determine whether these stores were necessary for avoidance . Baseline avoidance to GTP was seen at a thapsigargin concentration of 1,000 nM -LRB- Fig. 5b -RRB- . The IC50 of thapsigargin was approximately 1 nM . NO is another second messenger sometimes associated with tyrosine kinases -LSB- 5 , 17 -RSB- and often associated with calcium signaling -LSB- 5 , 18 -RSB- . In addition , NO has been found to be a critical second messenger in the Tetrahymena response to both ATP -LSB- 13 -RSB- and PACAP-38 -LSB- 18 -RSB- . To determine whether NO was involved in GTP signaling , we used the NOS inhibitors 1400W -LRB- Fig. 6a -RRB- and L-NMMA -LRB- Fig. 6b -RRB- . Both inhibitors blocked avoidance to 100 μM GTP-γ-S in Tetrahymena , with baseline avoidance being reached at 100 nM for 1400W -LRB- Fig. 6a -RRB- and 50 μM for L-NMMA -LRB- Fig. 6b -RRB- . The IC50 of 1400W was near 0.1 nM , whereas L-NMMA was approximately 3 μM . To confirm the activity of NOS in GTP avoidance , we used the Quantizyme NO assay to determine whether there was a difference in NO metabolism between control and GTP-stimulated cells . Cells stimulated with 8 mg/ml GTP had a 146 + / − 4.7 % increase in NO levels over control cells , consistent with the inhibitor studies -LRB- n = 3 -RRB- . In many cells , NO activates guanylyl cyclase , which then produces cGMP . Previous studies by Christensen et al. -LSB- 20 -RSB- described an NO-linked , possibly soluble guanylyl cyclase activity in T. thermophila . More recent studies , reviewed by Linder and Schultz -LSB- 20 -RSB- , described a membrane-associated and calcium-dependent guanylyl cyclase in these organisms . To determine whether any guanylyl cyclase isoform was involved in GTP avoidance , we used NS2028 , an inhibitor of guanylyl cyclase -LRB- Fig. 7a -RRB- , as well as Rp-8-bromo-cGMPs , a competitive inhibitor of cGMP -LRB- Fig. 7b -RRB- , in behavioral assays . NS2028 and Rp-8-bromo-cGMPs both blocked avoidance to 100 μM GTP-γ-S in Tetrahymena . Baseline avoidance was obtained at 10 nM NS2028 -LRB- Fig. 7a -RRB- and at 50 μM Rp-8-bromo-cGMPs -LRB- Fig. 7b -RRB- . The IC50 of NS2028 was approximately 0.05 nM and of Rp-8-bromo-cGMPs near 7 μM . To confirm these results biochemically , we performed an EIA assay to determine whether GTP exposure resulted in elevated cGMP levels within Tetrahymena . Exposure to 5 mg/ml GTP resulted in a 1,119 + / − 4.2 % increase above control cGMP levels -LRB- n ≥ 3 -RRB- , consistent with the hypothesis that GTP avoidance is linked to a guanylyl cyclase . Based on the data we collected , we put together a provisional model of GTP signaling -LRB- Fig. 8 -RRB- . In our model , GTP binds to a receptor , which activates tyrosine kinase while interacting with a membrane calcium channel , causing a depolarization . Phospholipase C is activated , generating IP3 , which allows calcium efflux from ER stores . The high cytosolic calcium interacts with some -LRB- unknown -RRB- isoform of NOS , guanylyl cyclase , and other calcium-binding proteins , ultimately resulting in ciliary reversal . Whereas some of the relationships between second messengers are still unknown , the model has been put together to stimulate thought and discussion and to make some order out of a complex pathway . Discussion The data we have compiled here are consistent with the hypothesis that the previously described GTP receptor -LSB- 1 -RSB- signals through a tyrosine kinase -LRB- Fig. 2 , 3 -RRB- . The GTP signaling pathway also involves phospholipase C -LRB- Fig. 4 -RRB- , intracellular calcium -LRB- Fig. 5 -RRB- , NOS -LRB- Fig. 6 -RRB- , and guanylyl cyclase -LRB- Fig. 7 -RRB- . The proposed GTP signaling pathway is outlined in Fig. 8 . Fig. 4The phospholipase C inhibitor 1 - -LRB- 6 - -LRB- 17β-3-methoxyestra-1,3,5 -LRB- 10 -RRB- - trien-17-yl -RRB- amino -RRB- hexyl -RRB- -1 H-pyrrole-2,5-dione -LRB- U73122 -RRB- blocks avoidance to 100 μM guanosine-5 ′ - O - -LRB- 3-thio -RRB- triphosphate -LRB- GTP-γ-S -RRB- in Tetrahymena . Baseline avoidance was reached at a U73122 concentration of 1 μM . The inhibition constant IC50 of this compound was near 0.001 μM . Data shown is mean + / − standard deviation -LRB- SD -RRB- of three or more trialsFig . 5Intracellular calcium is required for guanosine 5 ' - triphosphate -LRB- GTP -RRB- avoidance in Tetrahymena . a The membrane-permeable calcium chelator 1,2-bis - -LRB- o-Aminophenoxy -RRB- - ethane-N , N , N ' , N ' - tetraacetic acid , tetra -LRB- acetoxymethyl -RRB- ester -LRB- BAPTA-AM -RRB- blocks avoidance to 100 μM GTP-γ-S in Tetrahymena . Baseline avoidance was reached at a BAPTA-AM concentration of 5 μg/μl . The inhibition constant IC50 of this compound was near 0.5 μg/μl . Data shown is mean + / − standard deviation -LRB- SD -RRB- of three trials . b The endothelial reticulum -LRB- ER -RRB- calcium adenosine triphosphatase -LRB- ATPase -RRB- inhibitor thapsigargin also blocks avoidance to 100 μM guanosine-5 ′ - O - -LRB- 3-thio -RRB- triphosphate -LRB- GTP-γ-S -RRB- in Tetrahymena . . Baseline avoidance was reached at a thapsigargin concentration of 1,000 nM . The IC50 of this compound was near 1 nM . Data shown is mean + / − standard deviation -LRB- SD -RRB- of three or more trialsFig . 6Nitric oxide synthase -LRB- NOS -RRB- activity is required for guanosine 5 ' - triphosphate -LRB- GTP -RRB- avoidance in Tetrahymena . a The NOS inhibitor 1400W blocks avoidance to 100 μM guanosine-5 ′ - O - -LRB- 3-thio -RRB- triphosphate -LRB- GTP-γ-S -RRB- in Tetrahymena . Baseline avoidance was reached at a 1400W concentration of 100 nM . The inhibition constant IC50 of this compound was near 0.1 nM . Data shown is mean + / − standard deviation -LRB- SD -RRB- of three trials . b The NOS inhibitor NG-monomethyl-L-arginine -LRB- L-NMMA -RRB- also blocks avoidance to 100 μM GTP-γ-S in Tetrahymena . Baseline avoidance was reached at an L-NMMA concentration of 10 μM . The inhibition constant IC50 of this compound was near 3 μM . Data shown is mean + / − standard deviation -LRB- SD -RRB- of at least three trialsFig . 7Guanylyl cyclase activity is required for guanosine 5 ' - triphosphate -LRB- GTP -RRB- avoidance in Tetrahymena . a 4H-8-bromo-1,2,4-oxadiazolo -LRB- 3,4-d -RRB- benz -LRB- b -RRB- -LRB- 1,4 -RRB- oxazin-1-one -LRB- NS2028 -RRB- , an inhibitor of guanylyl cyclase , blocked avoidance to 100 μM guanosine-5 ′ - O - -LRB- 3-thio -RRB- triphosphate -LRB- GTP-γ-S -RRB- in Tetrahymena . Baseline avoidance was obtained at 10 nM . The inhibition constant IC50 of NS2028 was approximately 0.05 nM . Data shown is mean + standard deviation -LRB- SD -RRB- of three trials . b Rp-8-Bromoguanosine-3 ′ ,5 ′ - cyclic monophosphorothioate -LRB- Rp-8-Br-cGMPs -RRB- , a competitive inhibitor of guanosine 3 ′ 3 ; 5 ′ monophosphate -LRB- cGMP -RRB- , also blocked avoidance to 100 μM GTP-γ-S in Tetrahymena . Baseline avoidance was obtained at 50 μM . The IC50 of Rp-8-Br-cGMPs was approximately 7 μM . Data shown is mean + standard deviation -LRB- SD -RRB- of three or more trialsFig . 8Current model of guanosine 5 ' - triphosphate -LRB- GTP -RRB- signaling . Based on data obtained in this experiment , GTP appears to signal through a receptor linked to a tyrosine kinase . Phospholipase C , intracellular calcium release , nitric oxide -LRB- NO -RRB- production , and guanylyl cyclase are all involved in GTP avoidance . Some of the relationships between pathway components remain unknown . This model is based on the data presented in this paper , along with data obtained from the literature -LSB- 26 , 32 , 33 -RSB- GTP is the first chemorepellent we have studied that does not signal through a G-protein-linked receptor , unlike ATP -LSB- 13 -RSB- and PACAP -LSB- 18 , 21 -RSB- . In contrast to other repellents , GTP signaling is unaffected by either GDP-β-S or pertussis toxin -LRB- unpublished data -RRB- . GTP is also the only one of these three repellents whose signaling is eliminated by genistein -LRB- Fig. 2 ; see `` Results '' -RRB- . Previously published cross-adaptation data -LSB- 2 -RSB- indicated that GTP and ATP signal through different receptors . These data are consistent with the previously published literature and add the observation that both receptors and second-messenger pathways for these two repellents appear to be different . Multiple target proteins are phosphorylated in response to GTP exposure -LRB- Fig. 3d -RRB- , yet the proteins remain unidentified except for molecular weight . Phosphotyrosine immunolocalization does not help us identify these proteins ; however , the ciliary staining -LRB- Fig. 3b -RRB- opens up the possibility of direct interaction between tyrosine kinase activity and ciliary reversal . This is worthy of note , as Christensen et al. -LSB- 22 -RSB- report a similar increase in ciliary phosphotyrosine labeling after exposure to the chemoattractant insulin . However , in their report , the main protein that was phosphorylated was a 66 kDa protein , which they discovered was homologous to the insulin receptor . In the case of GTP exposure , we saw no increase in labeling of the 66 kDa protein -LRB- Fig. 3d -RRB- . Taken together , these data raise the possibility that there are multiple tyrosine kinase pathways that act on cilia to increase forward swim speed in the case of a chemoattractant , or to cause ciliary reversal in the case of a chemorepellent . Becausxe there were multiple phosphotyrosine bands in our Western blot -LRB- Fig. 3d -RRB- and ciliary as well as cytosolic staining in our immunofluorescence -LRB- Fig. 3b -RRB- , we see that GTP exposure has both ciliary and cytosolic effects . Ciliary reversal is seen immediately upon GTP exposure ; however , other effects of GTP exposure , such as mitosis , are seen hours after GTP exposure -LSB- 4 -RSB- . Perhaps the cytosolic target proteins are involved in other , longer-term effects of GTP , such as mitosis . Many tyrosine kinase pathways involve phospholipase C . In various Tetrahymena species , evidence for phospholipase C involvement has been seen in stomatin-induced differentiation -LSB- 24 -RSB- and in commitment to cell survival or death -LSB- 25 -RSB- . Studies by Leondaritis and Galanopoulou -LSB- 26 -RSB- suggest that Tetrahymena possess a functional phosphatidylinositol signaling system that is similar to higher eukaryotes . Indeed , in our previous studies of PACAP -LSB- 10 -RSB- and ATP -LSB- 13 -RSB- , phospholipase C involvement is also implicated . In our current study , we found that U73122 effectively eliminated GTP avoidance -LRB- Fig. 4 -RRB- , indicating that phospholipase C signaling is involved in behavioral avoidance . However , our calphostin C data -LRB- see `` Results '' -RRB- indicate that PKC , a common downstream target of phospholipase C , is not required for GTP avoidance . IP3 , a second messenger generated by phospholipase C , is likely involved in GTP signaling . Although we could not test IP3 involvement directly using our behavioral assay , the fact that the ER calcium ATPase inhibitor thapsigargin eliminated GTP avoidance -LRB- Fig. 5b -RRB- is indirect evidence that IP3 may be binding to the ER and causing release of calcium from internal stores . Calcium is a ubiquitous signaling molecule and has been implicated in ciliary reversal in Tetrahymena . It was no surprise , then , that BAPTA-AM eliminated GTP avoidance -LRB- Fig. 5a -RRB- , as it has previously been shown to eliminate avoidance to PACAP -LSB- 18 -RSB- and ATP -LSB- 13 -RSB- . Calcium is likely to play multiple roles in signaling . One possible scenario is the direct interaction of calcium with ciliary motor proteins . For example , inner arm dynein 1 -LRB- I1 -RRB- is required for calcium-dependent ciliary reversals in T. thermophila -LSB- 27 -RSB- . In this study , a `` calcium sensor , '' which interacts with I1 , was postulated . This calcium sensor protein remains to be isolated . Intracellular calcium may also play other , less direct , roles in GTP avoidance . Indeed , in the protozoan parasite Leishmania donovani , both NOS and guanylyl cyclase are activated by intracellular calcium -LSB- 28 -RSB- . Both NOS -LRB- Fig. 6 -RRB- and guanylyl cyclase -LRB- Fig. 7 -RRB- activity are required for GTP avoidance . Previous studies that implicate NOS and guanylyl cyclase in Tetrahymena -LSB- 13 , 18 , 19 -RSB- do not indicate the type of NOS or guanylyl cyclases involved , though Christensen et al. -LSB- 19 -RSB- give evidence for a soluble guanylyl cyclase . In L. donovani , there is evidence for a constitutive , neuronal-type NOS that then activates a soluble guanylyl cyclase -LSB- 28 -RSB- . Both of these enzymes are activated by intracellular calcium . Our data would be consistent with a similar mechanism in Tetrahymena , as proposed in our model of GTP signaling -LRB- Fig. 8 -RRB- . However , neither a soluble guanylyl cyclase nor an nNOS isoform have been purified from Tetrahymena to date . Calcium-binding proteins , such as calmodulin , may also be involved in GTP signaling in Tetrahymena , either by interacting with enzymes that generate second messengers or by interacting with the molecular motors responsible for ciliary reversal . Previous studies -LSB- 29 , 30 -RSB- indicate that calmodulin activates a ciliary guanylyl cyclase in this organism . Our studies leave open the possibility that this cyclase , activated by calcium/calmodulin , is involved in GTP signaling . In addition to this enzyme , Hirano-Ohnishi and Watanabe -LSB- 31 -RSB- reported a calmodulin-dependent phosphorylation of ciliary beta tubulin in Tetrahymena , which they believe to be involved in ciliary reversal . Elongation factor 1α , which functions in protein translation , also interacts with ciliary calmodulin -LSB- 32 -RSB- . Whereas this would not necessarily function in ciliary reversal , it may modulate some of the longer-term effects of GTP . Finally , Ueno et al. -LSB- 33 -RSB- report that ciliary calmodulin interacts with proteins that are homologues of radial spoke proteins in Chlamydomonas , further implicating calmodulin directly in ciliary reversal . We have added some of these proteins to our model of GTP signaling -LRB- Fig. 8 -RRB- , though there are doubtless more proteins to be discovered . In this study , we used behavioral assays and pharmacological inhibitors to explore the intracellular mechanisms employed in GTP signaling in T. thermophila . Because pharmacological inhibitors often have unknown side effects , we used multiple inhibitors when available . Some of these inhibitors had to be used at higher concentrations in our study than have been previously published for mammalian cells . Perhaps this is because of the differences in the structure of the Tetrahymena proteins compared with their mammalian counterparts . Recognizing the potential pitfalls of relying solely upon pharmacological data , we supplemented behavioral assays with other kinds of tests , such as Western blots , immunofluorescence , and EIA when available . In all cases , the additional tests confirmed the data we originally obtained using pharmacological inhibitors . Taken together , our data indicate that a number of enzymes are involved in GTP signaling . However , further experimentation is needed to discern the interrelationships between enzymes in the pathway and to answer the myriad questions that remain . For example , are guanylyl cyclase and NOS truly activated by calcium in Tetrahymena ? Would calcium chelation abolish their activity ? Is there a soluble guanylyl cyclase in Tetrahymena ? What NOS isoforms do Tetrahymena possess ? Is tyrosine phosphorylation required for either NOS or guanylyl cyclase activity ? Are any additional proteins , particularly ciliary proteins , involved in GTP signaling ? Are all of the components of the pathway located in the cilia ? These and many other questions remain to be answered . One question we have begun to answer is whether intracellular calcium is required for tyrosine kinase activity . Recent immunofluorescence shows no difference in phosphotyrosine levels between GTP-treated cells and GTP-treated cells that had been pretreated with BAPTA-AM -LRB- unpublished data -RRB- . Similarly , calcineurin -LRB- PP2B -RRB- does not appear to be involved in GTP signaling , as ascomycin treatment had no effect on GTP avoidance -LRB- unpublished data -RRB- . Endothall , a PP2A inhibitor , did inhibit avoidance -LRB- unpublished observation -RRB- , consistent with observations by Deckman and Pennock -LSB- 33 -RSB- that PP2A is involved in ciliary reversal . Further experimentation will help us determine the interrelationship of pathway components and better establish the mechanism by which chemorepellents signal in Tetrahymena .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

extracellular nucleotide
purine metabolism