Life Sciences, Vol . 24, pp . 1555-1566 Printed in the U .S .A .
Pergaaon Press
ASCORBIC ACID MODULATION OF SPLENIC CELL CYCLIC GMP METABOLISM Mari R. Haddoz, Janis H . Stepheneon, Mary E . Mosey, David B. Glase, James G . White, Beinah Holmen-Gray, and Nelson D. Goldberg Departments of Pharmacology, Laboratory Medicine and Pathology University of Mi~eaota, Minneapolis, Minnesota 55455 (Received in final form November 22, 1978) Summary Chronic aecorbate deprivation of guinea pigs decreased splenic cell cyclic GMP levels (80Z) ; aecorbate (1 ~ addition to these cells in vitro restored the cellular concentration to control levels . Spleaic cells from non-scorbutic animals also exhibited increases in cyclic GMP levels in response to ezogenoue aecorbate whereas thiol reducing agents diainished cellular cyclic GMP concentration. Agents that inhibit the propagation of free radicals prevented this cellular effect of aecorbate while agents known to interfere with or promote H202 production had no effect . Guanylate cyclase activity in cell lysates increased after treatment of intact cells with aecorbate; dithiothreitol reversed this effect . Ascorbate also enhanced guaaylate cyclase activity in cell lysates. The results suggest that ozidizing aquivalente in the form of the monoanionic free radical of aecorbate alter cyclic GMP metabolism is these cells by activating guanylate cyclase via a mechanism involving oxidation of a cyclase-related component. Ascorbic acid can promote increases in the concentration of cyclic 3',5'guanoeine monophoephate (cyclic GMP, cGMP) when added to suspensions of washed human platelets (1,2), white blood cells (3), or to segments of umbilical artery (4) . The mechanism by which this vitamin influences cGMP metabolism is unknown . Cellular events involving oxidation and reduction have been suggested to represent a general mechanism for the control of cGMP metabolism, specifically This concept ie through the modulation of guanylate cyclase activity (5-8) . supported by the corresponding oxidative-reductive related changes that can be demonstrated in intact epleaic cell cGMP metabolism and in the activity of Additional direct evidence of guaaylate cyclase after cell disruption (7) . the soluble guanylate cyclase activation by oxidative processes has appeared : enzymes from lung (9,10), platelets (11,12) and splenic cells (6,7) undergo a spontaneous activation in room air which can be suppressed by DTT or a N2 atmosphere ; substances such as NaNg, NHgOH, and nitrop :veside, which can generate ozidizing equivalents in the form of nitroxidae, are potent activators of the cyclase (5,13,14) ; carcinogenic nitrosamines which readily generate free radicals stimulate the enzyme is a manger suppressible by antiozidaata (15-17) ; and hydrozyl free radicals have been postulated to serve as an activating species of ozidant for guanylate cyclase (lg) .
0024-3205/79/171555-1202 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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Although the involvement of ascorbate in the modulation of cellular events has not yet been clearly defined, it is recognized that the vitamin participates in reversible oxidation-reduction reactions which are thought to relate to its physiological function . Since redoa-related processes appear to underlie a mechanism of regulating splenic cell cGMP metabolism, the possibility that ascorbate may represent one of the redox components in such a process was investigated . In this report it is shown that vitamin C deficiency lowers splenic cell cGMP levels while ascorbate restores or increases cGMP concentration. Characteristics of the vitamin C-induced increase in apleaic cells are presented which indicate that ascorbate may exert its effect on cGMP metabolism through as activation of guanylate cyclase by a process involving oxidation . Materials and Methods Control and scorbutogenic guinea pig diets ware obtained from Nutritional Biochemical Co ., and dehydroascorbic acid was purchased from ICN and Chemical Procurement Laboratories . All other test ants were obtained from Sigma . Cyclic [3 H]~, [14 C]ascorbic acid and Na12 I were purchased from New England Nuclear. Antibodies against cGMP were raised in goats, and the antiserum to CAMP was a gift from Dr . A . L . Steiner . 5,8,11,14 Eicosatetraynoic acid was a gift from Hoffma~-La Roche, Inc . Ascorbic acid, sodium ascorbate, dehydroascorbic acid and 2,3-diketogulosic acid were prepared as previously described (7) . Sílica gel thin-layer chromatography and differential dinitrophenylhydrazine analysis showed the ascorbic acid and the 2,3-diketogulonic acid solutions to be over 98X pure, and the dehydroascorbic acid to be 90X pure, containing 2,3~iketogulonic acid . Scurvy was induced in guinea pigs by the omission of ascorbic acid from the diet of the scorbutic group . The average intake of ascorbic acid by an animal in the control group was 50-60 mg/day . Animals on the scorbutic diet exhibíted all the classical symptoms of vitamin C deficiency (19) . At the time of sacrifice, the average body weights of control and scorbutic animals were 122X and 88X of their pre-experimental body weights, respectively . Animals were sacrificed 22-28 days after initiation of experimental diets when ascorbic acid determinations on liver samples showed the level of the vitamin in the scorbutic group was leas than 5X of the control group . Heart, liver, lung, spleen, and adrenal glands were excised and rapidly frozen by lmme reion in liquid nitrogen . Platelets were isolated from whole blood (12) . Enriched populations of peritoneal polymorphonuclear leukocytes or macrophages were obtained after peritoneal casein challenge (1 or 2 days, respectively) . Cell preparations were identified as 70X enriched by morphologic criteria . Splenic cell suspensions were prepared as previously described (7) and suspended in RPMI 1640 containing 25 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7 .4 . Contamination by platelets was determined to be insufficient to contribute significantly to the total cellular level of cyclic nucleotide measurable under the conditions employed . In experiments involving intact cells, viability of the aplenic cells was confirmed by trypan blue exclusion before incubation at 37 ° fot 30 min prior to the addition of agents to be tested . The incubations were terminated by the addition of HC104 to a final concentration of 1 N. Cyclic nucleotides were extracted, partially purified and assayed as previously described (7) . Guanylate cyclase activity was measured by a modification (7) of the method of Rimura and Murad (20), and phoephodiesterase by the method of O'Dea et al . (21) . Tissue ascorbic acid, dehydroascorbic acid,
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and 2,3-dikatogulonic acid, extracted with SnC12-HPOg, were determined by the differential dinitrophenylhydraziae method of Roe et al . (22) . Parity of stock solutions and chemical interconversione or catabolism of ascorbate or its metabolites during teat reactions was monitored by thin-layer chromatography (7) . Results Maintaining guinea pigs on a scorbutogenic diet did not significantly alter cQ~ levels of plasma, adrenals, platelets, lung, liver, heart, peritoneal polymorphoauclear leukocytes or macrophages compared to control animals. The spleen was the only organ e:amiaed in which ascorbate deficiency was reThe splenic concentration lated to a detectable alteration in cC~ metabolism . of cGMP was decreased from 150 t 10 fmoles/mg protein (n - 8) in control animals to 90 t 20 fmoles/n+g protein (n ~ 7) in scorbutic animals . Ascorbate 1 .78 levels in the spleens from scorbutic animals were strikingly diminished : vs . 0 .02 pg ascorbate/mg protein in the control and scorbutic spleens, respectively .
FIGURE 1 (A) Time dependence of the ascorbate-promoted increase in cGMP in normal and scorbutic guinea pig splenic cells . Spleaic cells prepared from normal (closed circles) or scorbutic (open circles) animals were incubated (37°) for the times indicated in the presence of 1 mM sodium ascorbate . (B) Concentration dependence of the ascorbate promoted increase is splenic cell eGMP concentration. Cells prepared from spleens of non-scorbutic guinea pigs were incubated (37 °) for 1 min in the presence of the indicated concentration of ascorbate. INSET: Splenic cell cGMP levels are shown as a function of the total ascorbate concentration present (i .e ., sum of endogenous plus exogenous) .
The cGMP concentration in isolated splenic cells prepared froa scorbutic animals was only 20x of the level is calls from non-scorbutic animals (Fig . lA). Addition of 1 mM sodium ascorbate, the concentration of the vitamin determined to be present in the non-scorbutic tissue, to the scorbutic cells restored the cGMP content to the level present is control cells within 30 sec . The increase ~woae maintained for at least 30 min . This rapid restoration of the intracellular tGMP concentration upon ascorbate addition to isolated cells suggnste that the cyclic nucleotide decrease occurring in the scorbutic animals ís a primary result of the vitamin deprivation and not a secondary consequence of other aepette of scorbutogenasis . The addition of vitamin C (1 ~ to tells from
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non-scorbutic animals also promoted an elevation in cGMP concentration. The temporal characteríatica of the ascorbate-induced cGMP increase were identical in cells from normal and acorbutic guinea pigs (Fig . lA) . Sodium ascorbate or ascorbic acid were equally effective. Figure 1B shows that the increase in aplenic cell cGMP is directly rQlated to the level of ascorbate added to the cells in a concentration range of the vitamin normally present in the spleen . An increase was demonstrable after the addition of as little as 0 .01 mM ascorbate . A maximal level of cGMP, 3-fold higher than basal, was achieved upon the addition of 10 mM ascorbate; higher concentrations of the vitamin promoted no further increase . Ia the inset of Figure 1B, the cellular cGMP concentration is plotted against the total ascorbate present (i .e ., calculated se the sum of the endogenous plus exogenously added vitamin C) . The concentration of cGMP representative of zero ascorbate was assumed to be the concentration of the cyclic nucleotide in cells from acorbutic cells which were 98 .9X depleted of ascorbate (i .e ., from Fig. lA) . According to this representation of the data as much as a 6-fold elevation in the cGMP levels (from 20 to 120 fmolea/10~ cells) can be attributed to ascorbate . The cGMP level corresponding to the normal level of cellular ascorbate (1 mt~ lien on the linear portion of the concentration curve. The concentration of ascorbate required to achieve 50X of the maximum cGMP level is 1 .1 mM . Therefore, a small change in the basal level of the vitamin could theoretically promote a relatively large alteration in cG~ concentration. A Ascorbate produced no significant change in the cAMP levels (Fig . 1B) . metabolite and chemical degradation product (23) of vitamin C, 2,3-dilcetogulonic acid, did not change the concentration of either cyclic nucleotide .
Minutes
FIGURE 2 Effect of ascorbate, dehydroascorbate, cysteine and DTT oa cGMP levels in guinea pig aplenic cells . Cell suspensions were incubated is the presence of a 1 mM concentration of the agent tested for the times indicated . The values shown are means of triplicate determinations that do not differ by more than 12X .
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Ascorbic Acid and Splenic Cell c-(7~
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The biological effects of ascorbate are ordinarily attributed to its reducing potential . However, when the effect of ascorbate oa eplenic cell cGMP metabolism was compared to the effect of two other agents with reducing potential, cyateine ~ad dithiothreitol, both were shown to have an effect opposite to that of ascorbate (Fig . 2) . The thiol reductaats caused a timedependeat decrease of 70 to 90x in the cellular cGMP content. In contrast, dehydroaecorbíc acid, the form of vitamin C possessing ozidizing potential, caused an enhanced accumulation of cGMP similar to that promoted by ascorbate (Fig . 2) (7) . No significant change was observed is the cAt~ levels upon the addition of say of the agenta tented . It appears, therefore, that the ascorbate-induced increase in eplenic cell cGl~ is more likely the consequence of an oxidative than a reductive process. One possible mechanism for generating oxidative equivalents from ascorbate is through its comrereioa to the deh~dro form, This possibility was tested by determining the metabolic fate of [ 4C]aacorbic acid (1 mt~ after its addition to the cell suspensions. There was no conversion of ascorbate to dehydroascorbate (i,e ., based on a minimum detectable level of 0 .01 mM dehydroascorbate) after 30 sec or 2 min of incubation when cGMP levels were elevated substantially in the cells .
au-:~,.aon~r
PBS
5 mll Aec.
1 mM DTT
FIGURE 3 Stability and reversibility of the ascorbate- and thiol reductant-promoted changes in eplenic cell cGMP concentration. Splenic cells were preincubated at a density of 2 a 10 6 cells/ ml phosphate-buffered saline (PBS), pH 7.4, for 5 min at 37 ° is the presence of 5 mK ascorbate or 1 mM DTT as indicated . The cells were sadimented by centrifugation for 1 min at 2000 rpm sad resuepèndzd in the original volume of PBS . The cells were either extracted immediately (solid bars) or incubated for as additional 5 min ín PBS, ascorbate, or DTT as indicated before extraction .
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The stability and reversibility of the aecorbate-induced increase in cellular cGMP was examined (Fig . 3) . The increase in Splenic cell cG!!P persisted after removal of aecorbate from the media by sedimentation and resuapensíon of the cells is aecorbate-free media . This elevation in eplenic call cGMP con centration is reversed by the addition of the thiol-containing reducing agent, dithiothreitol, to the resuspeaded cells (Fig . 3) . Furthermore, the effect of dithiothreitol to diminish cGt~ apleaic cell levels, which also persists after removal of the reductiot from the media, ie reversed upon the addition of aecorbate (Fig . 3) . These results favor stable changes induced through processes involving oxidation (i .e ., aecorbate) and reduction (i .e ., dithiothreitol) . Since the aecorbate-induced change in cGMP steady-state levels in the intact cell was relatively stable, it seemed likely that a cellular component responsible for the alteration in cGMP metabolism may have undergone a corres pondingly stable change as a result of vitamin C action . This was tested by comparing guanylate cyclase activity is lysates from cells that had or had not been exposed to aecorbate . Figure 4 illustrates that disrupted cells previously ezpoaed to ascorbate(but resuepended in aecorbate-free media before lysis) exhibit a 2- to 3-fold enhanced rate of cGMP generation . The more active guanylate cyclase from aecorbate-treated cells could be restored to the basal state of activity when the thiol reductant,dithiothreitol,was added to the reaction mixture 5 min after its initiation . Also shown in Figure 4 is the previously described (7) spontaneous activation of splenic cell guanylate cyclase that occurs during the course of the reaction and the effect that dithiothreitol addition to cells before disruption has to prevent it .
1000
500 F N
s~
ón
C7
100 5
10 Minutes
15
FIGURE 4
Stability of changes inducible in guaaylate cyclase activity upon exposure of intact cells to aecorbate . Intact eplenic cells suspended in PBS at 5 a 10 6 cells/ml were incubated (5 min, 30 ° ) in the presence of 5 mM aecorbate (0), 5 mM DTT (D) or without either of these agents (O) . The media containing the effectors was removed by sedimentation of the cells (2000 rpm, 75 sec) . Cello were resuspended at a density of 200 z 10 6 cells/ml of PBS and sonicated . Guanylate cyclase activity was determined with aliquots of cell lysates pretreated se described . The reversibility of the secorbate-enhanced rate of cGMP generation was examined by the addition of 1 mM DTT to the ongoing cyclase reaction at 5 min and assaying activity at the subsequent times indicated ( ") .
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Ascorbic Acid and Splenic Cell c-G1~
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In .two eaparismata conducted to determine whether soluble or particulate guaaylate cyclaee activity was activated upon exposure of cells to ascorbate, it was found that the enzyme activity in both fractions was elevated 50-SOX even after 60 min of ceatrífugation (105,000 x g) to obtain the fractions. The effectiveness of secorbate addition to intact cells to promote an apparently stable activation of guanylate cyclase prompted ezperímente to examine the ability of the vitamin to affect the activity of guanylate cyclase after cell disruption . In several eaperimeata where this was tested, little (15-20X) or no effect of ascorbate (0 .1-5 ~ was detected when freshly disrupted eonicates were analyzed (24) . However, is more recent ezperiments is which the lyaates were incubated at 0 ° before the addition of ascorbate and meaeuremeat of guanylate cyclase activity, ascorbate could be shown to produce increasingly greater activation of the enzyme with increasing time of preincubation; ascorbate addition more than doubled the activity after a 60~min preincubatioa of the lysate (Table 1) . The enzyme activity was unaffected by preincubation of the extract at 4° . The reaaon for the greater demonstrable effect of the vitamin upon preincubation of the lyeate hoe not been established . Activation of guaaylate cyclase by ascorbate also hoe bean reported by Liaag and Sector (25) with the soluble enzyme from rabbit renal tissue . It is noteworthy that in spite of the reducing potential of ascorbate, it does not exhibit the properties of the thiol-containing reductants to prevent the apoataneoue activation of the soluble enzyme from either fresh or preincubated lysatea (data not shown) . This may result from the lower reducing potential of the vitamin or reflect a requirement for a aulfhydryl-containing reductaat. Table 1 ASCORBATE STIMULABILITY OF SOLIIBLE SPLENIC CELL GUANYLATE CYCLASE WITS PREINCUBATION OF CELL LYSATE Time of Preincubation_
No Additions DTT Aacorbate (Qmol z m~ prot -l a 10 mia-1 ) -
0
319 t 10
5
295 t 52
15
208 t
X Activation by Aacorbate
394 t 20
124
_-_
479 t 27
163
306 t ib
---
537 t 39
175
30
321 t 23
---
59o t 14
184
60
290 t 25
652 t 12
224
0
200 t 32
Soluble spleaic cell extract was assayed with or without 5 mM ascorbate or Data represent 5 mM DTT after preincubatíoa at 4° for the times indicated . the means of triplicate determinations f SEM . The phoaphodiesteraee activity of disrupted apleaic cell suspenaioas also When the activity of thin was tested as a possible site of ascorbate action . enzyme was determined at substrate concentrations of cG?~ approximating the -e M) and at tw concentrations which Rm concentration of thin enzyme (1 z 10 represent pore physiological levels of the cyclic nucleotide in this tissue (1 : 10 -~ M and 1 a 10- M), there was no significant effect of ascorbate (0 .5 to 10 mM) on the activity of phosphodiesterase .
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Some characteristics of the process by which ascorbate influences cGMP metabolism, presumably through the activation of guanglate cyclase, were examined by testing the effect of agents with defined chemical actions to alter the ascorbate-induced cGMP response . The addition of EGTA (1 mM) to the Splenic cell suspensions, which resulted in a 15X lowering of the basal level of cGMP, did not impair the ability of ascorbate to increase cC~ . This is consistent with the Cam-independence of the effect of the oxidant periodate to elevate cGMP levels in these cells (5) and with the cationindependence of the ascorbate effect demonstrated in umbilical artery (4) . Aacorbate can participate in a non-enzymic, metal-catalyzed reaction with molecular oxygen which results is the generation of H20y (26) . Catalans (8 to 32 mg/ml of cell suspension), which would be expected to degrade any HpOp produced in the media, did not suppress the ascorbate-promoted increase in cellular cGMP accumulation . 1,2,4 Aminotriazole (0 .1, 1 .0 and 100 ml~ , which can enhance H20p-promoted events by inhibiting intracellular catalase act~.vity (27), also was without effect . The cyclo-oxygeaase pathway of fatty acid metabolism has been implicated in the ascorbate-promoted increase in cGMP in platelets (2) . However, preincubatioa of splenic cells (15 min, 37 ° ) with the cyclo-oxygenase inhibitors, aspirin (50 u~, indomethacin (50 ut~ or 5,8,11,14 eicosatetraynoic acid (50 uI~ (all of which lowered the basal cGMP concentration approximately 50X) had no significant effect on the magnitude of the increase in cGMP promoted by vitamin C . Diethyldithiocarbamic acid (DDC) (0 .05 and 0 .5 mM), a chelator with high affinity for transition metals and an effective free radical trapping agent (28), completely blocked the effect of ascorbate to elevate cGMP while lowering the basal concentrations 30-50X . The hindered phenolic anti-oxidants, 3t~utyl4-methyloxyphenol (BHA) and 4-methgl-2-6-di-t-butylphenol (BHT) also blocked the ascorbate-induced elevation in cGMP . These free radical scavengers had no effect on the basal cyclic nucleotide level . Discussion The relationship between ascorbic acid and splenic cell cGMP steadystate levels indicated by these results may have a two-fold significance . On one hand, it helps to further define a possible oxidative-reductive mechanism that may underly the cellular regulation of cG1+IP biosynthesis ; and, on the other, it identifies a pathway of splenic cell metabolism that may be regulated, at least in part, by vitamin C . That ascorbic acid acts as a physiological effector of cGt~ metabolism in these cells ie strongly suggested by the striking lowering (approximately 80X) of splenic cell cGMP concentration upon chronic vitamin C deprivation . Restoration of depleted levels of cGMP in isolated cells from scorbutic animals by concentrations of exogenous ascorbate that approximate the levels found in normal cells further implicates the vitamin as a possible natural cellular effector of cG[~IP metabolism is these cells . The Selectivity of vitamin C deprivation to affect the metabolism of this cyclic nucleotide in only splenic cells (and spleen) from among n~erous other cell types and tissues examined indicates that a relationship between vitamin C and cGMP metabolism of physiological significance may be restricted to only certain cell types . The effect of higher concentrations of exogenous ascorbic acid to promote cGl+lP accumulation ín umbilical artery (4), or in platelets (1,2,29) and macrophages (3) which showed no change in cGMP levels upon vitamin C deprivation, may sot be representative of the same relationship between
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Ascorbic Acid and Splenic Cell c-GIS
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vitamin C and cGMP uncovered in splenic celle . Ordinarily, ascorbic acid ie viewed as a substance which participates in redoz-related reactions because of its reducing potential, but its effect to elevate splenic cell c(~ paralleled the effect of oaidaata and was opposite to the effect of thíol reductaata . Thiol reductants also were effective in reversing the ascorbate-induced increase in cellular cGl~ . These results, combined with the recently reported evidence that guanylate cyclase from these cells can be activated by an apparent oxidative process (7), underscore the likelihood that the effects of ascorbate on splenic cell cGl~ metabolism result from the generation of oxidizing equivalents. Dehydroascorbíc acid can promote increases in Splenic cell c(~ steady-state levels sad activate the soluble and particulate forms of guanylate cyclase from these cells (7) . However, it is not likely that this metabolite represents the oaidiziag equivalent derived from ascorbate that affects cG~ metabolism in this system . Ten times more dehydroascorbate is required to elevate cG~iP levels in intact splenic cells sad to activate guanylate cyclase (7) in cell lysates than the concentrations of ascorbate effective in elevating cGMP levels in these cells . There was no detectable metabolism of dehydroascorbate to diketogulonic acid or to ascorbate in the intact or broken cell systems that would account for this greater requirement of dehydroascorbate . Furthermore, there ie no detectable conversion of ascorbate to dehydroascorbate during the time that as effect of the vitamin on cGMP metabolism in the intact cell is observable . Although the production of H20p can result upon interaction of ascorbate with ADP sad/or a transition metal (26), o~ddiziag equivalents in the form of Hp02 are not likely to be involved in the ascorbate-promoted elevation of cGt~ since catalane does not interfere with and 1,2,4 amisotriazole does not potentiate the effect ; also, HpOp (1-100 ~, in contrast to other oxidants (7,30), does not serve to elevate intact cell cGHP levels or to activate splenic cell guanylate cyclase. The results of the present studies also minimize the possibility of an involvement of Cam and the production of proataglaadine is the ascorbate effect on cGlíP metabolism in these cells. Schoepflin et al . (2) have reported that fatty acid cyclo-oaygenase inhibitors, auch as indomethacin, inhiHit ascorbate-induced cGi~ accumulation in platelets, but this was not found to be the case in the present studies nor with platelets in our earlier studies with ascorbate (1,29) . The basis for the disparate results are not known, but one difference besides the two different cell types employed is the two experiments was the use of the higher concentrations of ascorbate in the studies cited (2) . The transition metal requirement for the activation process, indicated by the inhibition by DDC, is similar to that previously shown for spontaneous activation of the soluble enzyme from lung (10) sad uterus (31) as well ae the activation of the enzyme from various sources by sodium azide (B) . The effectiveness of DDC as a free radical trapping agent ie, however, also recognized (28) and coupled with the observation that the ascorbate-induced elevation of splenic cell cGMP ie inhibited by the classical free radical scavengers, BHT and BHA, strongly suggests that free radical generation is involved in this action of ascorbate . In biological reactions is which ascorbate serves to promote oxidation which does not involve H20p production or conversion of the vitamin to its dehydro form (both of which seem unlikely is the present studies), it has bean postulated that the oaidiziag effector ie the labile, one electron oxidation product of ascorbate, moaodehydroascorbate (32-34) . Ascorbate oxidation, promoted either enzymically by ascorbate oaidase (35-37) or nonenzymically by comproportionation between ascorbate and
dehydroascorbate (38,39) or by transition metal catalysis (40) hen been shown to proceed according to the Michaelie formulation of a two-step oxidation which gives rise to a free radical intermediate (41) . The eaiatence of a
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Vol . 24, No . 17, 1979
semi-oxidized form of ascorbate which can serve as a relatively potent oxidant has been confirmed by electron spin resonance spectroscopy (37,42-46) . Guanylate cyclase activation by free radicals also has been suggested in reports on the activation of the enzyme by several nitroso-containing compounds (8, 15,17) . It also has been suggested that hydroxy-free radicals can activate guaaylate cyclase from several sources (18) . The direct evidence for a free radical mechanism of activation is not yet available, The demonstration that a relatively stable, more active form of the enzyme is measurable in lysates of intact cells treated with vitamin C indicates that the ascorbate-induced accumulation of cGMP in these cells results from an activation of guanylate cyclase . This is supported by the demonstration that ascorbate addition to the cell lysates also promotes enhanced guanylate cyclase activity although the undefined condition of preincubating the lysate (i .e ., before ascorbate addition) is required to maximize the effect . The effectiveness of the thiol reductiot, dithiothreitol, to convert the more active form of guanylate cyclase in lysates from ascorbate-treated cells to the less active basal state favors the view that ascorbate promotes guanylate cyclase activation in the intact cell by a process that involves oxidation of the enzyme or a closely associated regulatory component . These results are consistent with the concept that oxidation-reduction related processes represent one general mechanism by which cellular guanylate cyclase activity may be regulated and cellular steady-state levels of cGMP maintained or altered . Ackaawledgementa This research was supported by grants from the American Cancer Society (BC-166), the National Institutes of Health (NS-05979, NL-06314), and the Minnesota Leukemia Foundation . The preset address of Mari K . Haddox is Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724 . The present address of David B . Glass is Department of Pharmacology, School of Medicine, Emory University, Atlanta, Georgia 30322, Requests for reprints should be addressed to Dr . Nelson D . Goldberg, Department of Pharmacology, University of Minnesota, 105 Millard Hall, 435 Delaware Street, S .E ., Minneapolis, MN 55455 . Ref rences 1, 2, 3. 4. 5. 6.
7. 8.
N .D . GOLDBERG, M,R . HADDOX, S .E . NICOL, D .B . GLASS, C .H . SANFORD, F .A . RUEHL, Jr . and R . ESTENSEN, Adv . Cyclic Nucleotide Rea . _5 307-330 (1975) . G .S . SCHOEPFLIN, W . PICRETT, R .F . AUSTEN and E .J . GOETZL, J . Cyclic Nucleotide Res . _3 355-365 (1977) . J .A . SANDLER, J .I . GALLIN and M . VAUGHAN, J . Cell Biol . _67 480-484 (1975) . R .I . CLYMAN, A .S . BLACRSIN, V,C . MANGANIELLO and M . VAUGHAN, Pro c . Natl . Acad . Sci . USA _72 3883-3887 (1975) . M .R, HADDOR, L .T, FURCHT, S .R . GENTRY, M .E . MOSER, J .H . STEPHENSON and N .D . GOLDBERG, Nature _262 146-148 (1976) . M .K, HADDOX, J .H . STEPHENSON, M .E . MOSER, L .T . FURCHT, S .R . GENTRY and N .D . GOLDBERG, in Growth Kinetics and Biochemical Regulation of Normal and Malignant Cells (eds ., B . Drewinko and R .M . Humphrey) pp, 255-269, Williams and Wilkins, Baltimore (1977) . M .R . HADDOX, J .H, STEPHENSON, M .E . MOSER and N .D . GOLDBERG, J . Biol, Chem . 253 3143-3152 (1978) . C .K . MITTAL and F . MURAD, J . Cyclic Nucleotide Res . 3 381-392 (1977) .
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30 . 31 . 32 . 33 . 34 . 35 . 36 . 37 . 38 . 39 . 40 . 41 . 42 . 43 . 44 .
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T .D . CHRISMAN, D .L . GABBERS, M .A . PARKS and J .G . HARDMAN, J . Biol . Chew . 250 374-381 (1975) . A .A . WHITE, R .M . CRAWFORD, C .S . PATT and P .J . LAD, J . Biol . Chew. 251 7304-7312 (1976) . E . BOH!!S, R . JUNG and I . MECHLER, is Methode in Snzymolo®, Vol . 38 C (~de ., J .G . Hardman and B .W . O'Malley) pp . 199-202, Academic Preen, New York (1974) . D .B . GLASS, W . FREY, D .W . CARR and N .D . GOLDBERG, J . Biol . Chem . 252 1279-1285 (1977) . H . RIMURA, C .R . MITTAL and F . MURAD, J . Biol . Chew . _250 8015-8022 (1975) . S . ICATS~I, W .P . ARNOLD, C .R . MITTAL and F . MURAD, J . Cyclic Nucleotide Res . 3 25-35 (1977) . F .R . DeRUYERTIS and P .A . CRAVEN, Science _193 897-899 (1976) . F .R . DeRUBERTIS and P .A . CRAVEN, J . Biol . Chem . _251 4651-4658 (1976) . F .R . DeRUBERTIS and P .A . CRAVEN, J . Biol . Chew . _252 5804-5814 (1977) . C .R . MITTAL and F . MiJRAD, Proc . Natl . Acad . Sci . U3A 74 4360-4363 (1977) . M .E . REID, in The Vitamine (eda ., W .H . Sebrell and R .S . Harris) p . 269, Academic Press, New York (1954) . H . RIMIIRA and F . MURAD, J . Biol . Chew . _249 6910916 (1974) . R .F . O'DBA, M .R . HADDO% and N .D . GOLDBERG, J . Biol . Chem . _246 6183-6190 (1971) . J .H . ROE, M .B . MII.LS, M .J . OSSTERLING and C .M . DAMBON, J . Biol . Chen . _174 201-208 (1948) . P .G . DAYTON, F . EISENBERG and J .J . BúRNS, Arch . Biochem . Biophya . _81 111-118 (1959) . N .D . GOLDBERG, G . GRAFF, M .R . HADDOR, J .H . STEPHSNSON, D .B . GLASS and M .S . MOSER, Adv . Cyclic Nucleotide Res . 9 3143-3152 (1978) . C .T . LIANG and B . SACTOR, J . Cyclic Nucleotide Bee . _4 97-111 (1978) . C .R . DAWSON and R . TORUYAMA, Aun . N .Y . Acad . Sci . 9 2 212-222 (1961) . W .H . EVANS and M . RECHCIGL, Jr ., Biockim . Biophya . acta _148 243-250 (1967) . Y . ISHIMURA and 0 . HAYAISHI, J . Biol . Chew . 248 8610-8612 (1973) . N .D . GOLDBERG, M .R . HADDOR, S .E . NICOL, T .S .aCOTT, C .E . Z,EILIG and D .B . GLASS, is Proceedi a ICN-UCLA S osia on Molecular and Cellular Biology : Developmental Biology (ade ., D . Mchfahon and C .F . Foz pp . 440472, W .A . Benjamin Press, Menlo Park, California (1975) . G . GRAFF, J .H . STEPHENSON, D .B . GLASS, M .R . HADDO% and N .D . GOLDBERG, J . Biol . Chew. _253 (ia preen) (1978) . R .C . RRASRA, J .H . STBPHSNSON and N .D . GOLDBERG, Fed . Proc . _36 686 (1977) . W .D . WOSILAT, A . NASON and A . TERRELL, J . Biol . Chew . 206 271-282 (1954) . M . RSRN and S . RACER, Arch . Biochem . Biophya . _48 235-236 (1954) . H . ABASTEN, W. BERSTEN and Hj . STAUDINGER, Biochim . Biophya . Acta _27 598-608 (1958) . I . YAMAZARI and L .H . PIETTE, Biochim . Biophya . Acta 50 629 (1961) . Aj . STAUDINGER, R . KRISCH and S . LSONHAUSSR, Ann . N .Y. Acad . Sci . _92 195-207 (1961) . T . IYANAGI and I . YAMAZARI, Biochim . Biophya . Acta _172 370-381 (1969) . W . SCHINDER, W . WEIS and Hj . STAUDINGER, Biothun . Biophya . Acta _89 548-549 (1964) . W . SCHNEIDER and Hj . STAUDINGER, Biochim . Biophya . Acta _96 157-159 (1965) . A . WSISSBSRGSR and J .E . LuVALLS, Am . Chew . Soc . J . 66 700-705 (1944) . L . MICHAELIS, J . Biol . Chem . _96 703-715 (1932) . G . VonFOERSTER, W . WEIS and Hj . STAUDINGER, Juetue Liebigs Ann. . Chem . 690 166-169 (1965) . T . ONISHI, H . YAMAZARI, T . IYANAGI, T . NARAMURA and I . YAM"7~YT ~ Biochim . Biophya . Acta 172 357-369 (1969) . N . URI, in Autozidationand Aatio:idanta , Vol . I (ed ., W .O . Lundberg) pp . 133-169, Intarecience Preee, New York (1961) .
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L . REICH and S .S . STIVALA, Antoxidation of Hydrocarbons and Polyolefins , pp . 139-231, Dekker, New York (1969) . W .A . ERYOR, in Free Radicals is Biology (ed ., W .A . Pryor), pp . 1-49, Academic Presa, New York (1976) .
Pergaaon Press
ASCORBIC ACID MODULATION OF SPLENIC CELL CYCLIC GMP METABOLISM Mari R. Haddoz, Janis H . Stepheneon, Mary E . Mosey, David B. Glase, James G . White, Beinah Holmen-Gray, and Nelson D. Goldberg Departments of Pharmacology, Laboratory Medicine and Pathology University of Mi~eaota, Minneapolis, Minnesota 55455 (Received in final form November 22, 1978) Summary Chronic aecorbate deprivation of guinea pigs decreased splenic cell cyclic GMP levels (80Z) ; aecorbate (1 ~ addition to these cells in vitro restored the cellular concentration to control levels . Spleaic cells from non-scorbutic animals also exhibited increases in cyclic GMP levels in response to ezogenoue aecorbate whereas thiol reducing agents diainished cellular cyclic GMP concentration. Agents that inhibit the propagation of free radicals prevented this cellular effect of aecorbate while agents known to interfere with or promote H202 production had no effect . Guanylate cyclase activity in cell lysates increased after treatment of intact cells with aecorbate; dithiothreitol reversed this effect . Ascorbate also enhanced guaaylate cyclase activity in cell lysates. The results suggest that ozidizing aquivalente in the form of the monoanionic free radical of aecorbate alter cyclic GMP metabolism is these cells by activating guanylate cyclase via a mechanism involving oxidation of a cyclase-related component. Ascorbic acid can promote increases in the concentration of cyclic 3',5'guanoeine monophoephate (cyclic GMP, cGMP) when added to suspensions of washed human platelets (1,2), white blood cells (3), or to segments of umbilical artery (4) . The mechanism by which this vitamin influences cGMP metabolism is unknown . Cellular events involving oxidation and reduction have been suggested to represent a general mechanism for the control of cGMP metabolism, specifically This concept ie through the modulation of guanylate cyclase activity (5-8) . supported by the corresponding oxidative-reductive related changes that can be demonstrated in intact epleaic cell cGMP metabolism and in the activity of Additional direct evidence of guaaylate cyclase after cell disruption (7) . the soluble guanylate cyclase activation by oxidative processes has appeared : enzymes from lung (9,10), platelets (11,12) and splenic cells (6,7) undergo a spontaneous activation in room air which can be suppressed by DTT or a N2 atmosphere ; substances such as NaNg, NHgOH, and nitrop :veside, which can generate ozidizing equivalents in the form of nitroxidae, are potent activators of the cyclase (5,13,14) ; carcinogenic nitrosamines which readily generate free radicals stimulate the enzyme is a manger suppressible by antiozidaata (15-17) ; and hydrozyl free radicals have been postulated to serve as an activating species of ozidant for guanylate cyclase (lg) .
0024-3205/79/171555-1202 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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Ascorbic Acid and Splenic Cell c-Q4p
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Although the involvement of ascorbate in the modulation of cellular events has not yet been clearly defined, it is recognized that the vitamin participates in reversible oxidation-reduction reactions which are thought to relate to its physiological function . Since redoa-related processes appear to underlie a mechanism of regulating splenic cell cGMP metabolism, the possibility that ascorbate may represent one of the redox components in such a process was investigated . In this report it is shown that vitamin C deficiency lowers splenic cell cGMP levels while ascorbate restores or increases cGMP concentration. Characteristics of the vitamin C-induced increase in apleaic cells are presented which indicate that ascorbate may exert its effect on cGMP metabolism through as activation of guanylate cyclase by a process involving oxidation . Materials and Methods Control and scorbutogenic guinea pig diets ware obtained from Nutritional Biochemical Co ., and dehydroascorbic acid was purchased from ICN and Chemical Procurement Laboratories . All other test ants were obtained from Sigma . Cyclic [3 H]~, [14 C]ascorbic acid and Na12 I were purchased from New England Nuclear. Antibodies against cGMP were raised in goats, and the antiserum to CAMP was a gift from Dr . A . L . Steiner . 5,8,11,14 Eicosatetraynoic acid was a gift from Hoffma~-La Roche, Inc . Ascorbic acid, sodium ascorbate, dehydroascorbic acid and 2,3-diketogulosic acid were prepared as previously described (7) . Sílica gel thin-layer chromatography and differential dinitrophenylhydrazine analysis showed the ascorbic acid and the 2,3-diketogulonic acid solutions to be over 98X pure, and the dehydroascorbic acid to be 90X pure, containing 2,3~iketogulonic acid . Scurvy was induced in guinea pigs by the omission of ascorbic acid from the diet of the scorbutic group . The average intake of ascorbic acid by an animal in the control group was 50-60 mg/day . Animals on the scorbutic diet exhibíted all the classical symptoms of vitamin C deficiency (19) . At the time of sacrifice, the average body weights of control and scorbutic animals were 122X and 88X of their pre-experimental body weights, respectively . Animals were sacrificed 22-28 days after initiation of experimental diets when ascorbic acid determinations on liver samples showed the level of the vitamin in the scorbutic group was leas than 5X of the control group . Heart, liver, lung, spleen, and adrenal glands were excised and rapidly frozen by lmme reion in liquid nitrogen . Platelets were isolated from whole blood (12) . Enriched populations of peritoneal polymorphonuclear leukocytes or macrophages were obtained after peritoneal casein challenge (1 or 2 days, respectively) . Cell preparations were identified as 70X enriched by morphologic criteria . Splenic cell suspensions were prepared as previously described (7) and suspended in RPMI 1640 containing 25 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7 .4 . Contamination by platelets was determined to be insufficient to contribute significantly to the total cellular level of cyclic nucleotide measurable under the conditions employed . In experiments involving intact cells, viability of the aplenic cells was confirmed by trypan blue exclusion before incubation at 37 ° fot 30 min prior to the addition of agents to be tested . The incubations were terminated by the addition of HC104 to a final concentration of 1 N. Cyclic nucleotides were extracted, partially purified and assayed as previously described (7) . Guanylate cyclase activity was measured by a modification (7) of the method of Rimura and Murad (20), and phoephodiesterase by the method of O'Dea et al . (21) . Tissue ascorbic acid, dehydroascorbic acid,
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and 2,3-dikatogulonic acid, extracted with SnC12-HPOg, were determined by the differential dinitrophenylhydraziae method of Roe et al . (22) . Parity of stock solutions and chemical interconversione or catabolism of ascorbate or its metabolites during teat reactions was monitored by thin-layer chromatography (7) . Results Maintaining guinea pigs on a scorbutogenic diet did not significantly alter cQ~ levels of plasma, adrenals, platelets, lung, liver, heart, peritoneal polymorphoauclear leukocytes or macrophages compared to control animals. The spleen was the only organ e:amiaed in which ascorbate deficiency was reThe splenic concentration lated to a detectable alteration in cC~ metabolism . of cGMP was decreased from 150 t 10 fmoles/mg protein (n - 8) in control animals to 90 t 20 fmoles/n+g protein (n ~ 7) in scorbutic animals . Ascorbate 1 .78 levels in the spleens from scorbutic animals were strikingly diminished : vs . 0 .02 pg ascorbate/mg protein in the control and scorbutic spleens, respectively .
FIGURE 1 (A) Time dependence of the ascorbate-promoted increase in cGMP in normal and scorbutic guinea pig splenic cells . Spleaic cells prepared from normal (closed circles) or scorbutic (open circles) animals were incubated (37°) for the times indicated in the presence of 1 mM sodium ascorbate . (B) Concentration dependence of the ascorbate promoted increase is splenic cell eGMP concentration. Cells prepared from spleens of non-scorbutic guinea pigs were incubated (37 °) for 1 min in the presence of the indicated concentration of ascorbate. INSET: Splenic cell cGMP levels are shown as a function of the total ascorbate concentration present (i .e ., sum of endogenous plus exogenous) .
The cGMP concentration in isolated splenic cells prepared froa scorbutic animals was only 20x of the level is calls from non-scorbutic animals (Fig . lA). Addition of 1 mM sodium ascorbate, the concentration of the vitamin determined to be present in the non-scorbutic tissue, to the scorbutic cells restored the cGMP content to the level present is control cells within 30 sec . The increase ~woae maintained for at least 30 min . This rapid restoration of the intracellular tGMP concentration upon ascorbate addition to isolated cells suggnste that the cyclic nucleotide decrease occurring in the scorbutic animals ís a primary result of the vitamin deprivation and not a secondary consequence of other aepette of scorbutogenasis . The addition of vitamin C (1 ~ to tells from
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non-scorbutic animals also promoted an elevation in cGMP concentration. The temporal characteríatica of the ascorbate-induced cGMP increase were identical in cells from normal and acorbutic guinea pigs (Fig . lA) . Sodium ascorbate or ascorbic acid were equally effective. Figure 1B shows that the increase in aplenic cell cGMP is directly rQlated to the level of ascorbate added to the cells in a concentration range of the vitamin normally present in the spleen . An increase was demonstrable after the addition of as little as 0 .01 mM ascorbate . A maximal level of cGMP, 3-fold higher than basal, was achieved upon the addition of 10 mM ascorbate; higher concentrations of the vitamin promoted no further increase . Ia the inset of Figure 1B, the cellular cGMP concentration is plotted against the total ascorbate present (i .e ., calculated se the sum of the endogenous plus exogenously added vitamin C) . The concentration of cGMP representative of zero ascorbate was assumed to be the concentration of the cyclic nucleotide in cells from acorbutic cells which were 98 .9X depleted of ascorbate (i .e ., from Fig. lA) . According to this representation of the data as much as a 6-fold elevation in the cGMP levels (from 20 to 120 fmolea/10~ cells) can be attributed to ascorbate . The cGMP level corresponding to the normal level of cellular ascorbate (1 mt~ lien on the linear portion of the concentration curve. The concentration of ascorbate required to achieve 50X of the maximum cGMP level is 1 .1 mM . Therefore, a small change in the basal level of the vitamin could theoretically promote a relatively large alteration in cG~ concentration. A Ascorbate produced no significant change in the cAMP levels (Fig . 1B) . metabolite and chemical degradation product (23) of vitamin C, 2,3-dilcetogulonic acid, did not change the concentration of either cyclic nucleotide .
Minutes
FIGURE 2 Effect of ascorbate, dehydroascorbate, cysteine and DTT oa cGMP levels in guinea pig aplenic cells . Cell suspensions were incubated is the presence of a 1 mM concentration of the agent tested for the times indicated . The values shown are means of triplicate determinations that do not differ by more than 12X .
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Ascorbic Acid and Splenic Cell c-(7~
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The biological effects of ascorbate are ordinarily attributed to its reducing potential . However, when the effect of ascorbate oa eplenic cell cGMP metabolism was compared to the effect of two other agents with reducing potential, cyateine ~ad dithiothreitol, both were shown to have an effect opposite to that of ascorbate (Fig . 2) . The thiol reductaats caused a timedependeat decrease of 70 to 90x in the cellular cGMP content. In contrast, dehydroaecorbíc acid, the form of vitamin C possessing ozidizing potential, caused an enhanced accumulation of cGMP similar to that promoted by ascorbate (Fig . 2) (7) . No significant change was observed is the cAt~ levels upon the addition of say of the agenta tented . It appears, therefore, that the ascorbate-induced increase in eplenic cell cGl~ is more likely the consequence of an oxidative than a reductive process. One possible mechanism for generating oxidative equivalents from ascorbate is through its comrereioa to the deh~dro form, This possibility was tested by determining the metabolic fate of [ 4C]aacorbic acid (1 mt~ after its addition to the cell suspensions. There was no conversion of ascorbate to dehydroascorbate (i,e ., based on a minimum detectable level of 0 .01 mM dehydroascorbate) after 30 sec or 2 min of incubation when cGMP levels were elevated substantially in the cells .
au-:~,.aon~r
PBS
5 mll Aec.
1 mM DTT
FIGURE 3 Stability and reversibility of the ascorbate- and thiol reductant-promoted changes in eplenic cell cGMP concentration. Splenic cells were preincubated at a density of 2 a 10 6 cells/ ml phosphate-buffered saline (PBS), pH 7.4, for 5 min at 37 ° is the presence of 5 mK ascorbate or 1 mM DTT as indicated . The cells were sadimented by centrifugation for 1 min at 2000 rpm sad resuepèndzd in the original volume of PBS . The cells were either extracted immediately (solid bars) or incubated for as additional 5 min ín PBS, ascorbate, or DTT as indicated before extraction .
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The stability and reversibility of the aecorbate-induced increase in cellular cGMP was examined (Fig . 3) . The increase in Splenic cell cG!!P persisted after removal of aecorbate from the media by sedimentation and resuapensíon of the cells is aecorbate-free media . This elevation in eplenic call cGMP con centration is reversed by the addition of the thiol-containing reducing agent, dithiothreitol, to the resuspeaded cells (Fig . 3) . Furthermore, the effect of dithiothreitol to diminish cGt~ apleaic cell levels, which also persists after removal of the reductiot from the media, ie reversed upon the addition of aecorbate (Fig . 3) . These results favor stable changes induced through processes involving oxidation (i .e ., aecorbate) and reduction (i .e ., dithiothreitol) . Since the aecorbate-induced change in cGMP steady-state levels in the intact cell was relatively stable, it seemed likely that a cellular component responsible for the alteration in cGMP metabolism may have undergone a corres pondingly stable change as a result of vitamin C action . This was tested by comparing guanylate cyclase activity is lysates from cells that had or had not been exposed to aecorbate . Figure 4 illustrates that disrupted cells previously ezpoaed to ascorbate(but resuepended in aecorbate-free media before lysis) exhibit a 2- to 3-fold enhanced rate of cGMP generation . The more active guanylate cyclase from aecorbate-treated cells could be restored to the basal state of activity when the thiol reductant,dithiothreitol,was added to the reaction mixture 5 min after its initiation . Also shown in Figure 4 is the previously described (7) spontaneous activation of splenic cell guanylate cyclase that occurs during the course of the reaction and the effect that dithiothreitol addition to cells before disruption has to prevent it .
1000
500 F N
s~
ón
C7
100 5
10 Minutes
15
FIGURE 4
Stability of changes inducible in guaaylate cyclase activity upon exposure of intact cells to aecorbate . Intact eplenic cells suspended in PBS at 5 a 10 6 cells/ml were incubated (5 min, 30 ° ) in the presence of 5 mM aecorbate (0), 5 mM DTT (D) or without either of these agents (O) . The media containing the effectors was removed by sedimentation of the cells (2000 rpm, 75 sec) . Cello were resuspended at a density of 200 z 10 6 cells/ml of PBS and sonicated . Guanylate cyclase activity was determined with aliquots of cell lysates pretreated se described . The reversibility of the secorbate-enhanced rate of cGMP generation was examined by the addition of 1 mM DTT to the ongoing cyclase reaction at 5 min and assaying activity at the subsequent times indicated ( ") .
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Ascorbic Acid and Splenic Cell c-G1~
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In .two eaparismata conducted to determine whether soluble or particulate guaaylate cyclaee activity was activated upon exposure of cells to ascorbate, it was found that the enzyme activity in both fractions was elevated 50-SOX even after 60 min of ceatrífugation (105,000 x g) to obtain the fractions. The effectiveness of secorbate addition to intact cells to promote an apparently stable activation of guanylate cyclase prompted ezperímente to examine the ability of the vitamin to affect the activity of guanylate cyclase after cell disruption . In several eaperimeata where this was tested, little (15-20X) or no effect of ascorbate (0 .1-5 ~ was detected when freshly disrupted eonicates were analyzed (24) . However, is more recent ezperiments is which the lyaates were incubated at 0 ° before the addition of ascorbate and meaeuremeat of guanylate cyclase activity, ascorbate could be shown to produce increasingly greater activation of the enzyme with increasing time of preincubation; ascorbate addition more than doubled the activity after a 60~min preincubatioa of the lysate (Table 1) . The enzyme activity was unaffected by preincubation of the extract at 4° . The reaaon for the greater demonstrable effect of the vitamin upon preincubation of the lyeate hoe not been established . Activation of guaaylate cyclase by ascorbate also hoe bean reported by Liaag and Sector (25) with the soluble enzyme from rabbit renal tissue . It is noteworthy that in spite of the reducing potential of ascorbate, it does not exhibit the properties of the thiol-containing reductants to prevent the apoataneoue activation of the soluble enzyme from either fresh or preincubated lysatea (data not shown) . This may result from the lower reducing potential of the vitamin or reflect a requirement for a aulfhydryl-containing reductaat. Table 1 ASCORBATE STIMULABILITY OF SOLIIBLE SPLENIC CELL GUANYLATE CYCLASE WITS PREINCUBATION OF CELL LYSATE Time of Preincubation_
No Additions DTT Aacorbate (Qmol z m~ prot -l a 10 mia-1 ) -
0
319 t 10
5
295 t 52
15
208 t
X Activation by Aacorbate
394 t 20
124
_-_
479 t 27
163
306 t ib
---
537 t 39
175
30
321 t 23
---
59o t 14
184
60
290 t 25
652 t 12
224
0
200 t 32
Soluble spleaic cell extract was assayed with or without 5 mM ascorbate or Data represent 5 mM DTT after preincubatíoa at 4° for the times indicated . the means of triplicate determinations f SEM . The phoaphodiesteraee activity of disrupted apleaic cell suspenaioas also When the activity of thin was tested as a possible site of ascorbate action . enzyme was determined at substrate concentrations of cG?~ approximating the -e M) and at tw concentrations which Rm concentration of thin enzyme (1 z 10 represent pore physiological levels of the cyclic nucleotide in this tissue (1 : 10 -~ M and 1 a 10- M), there was no significant effect of ascorbate (0 .5 to 10 mM) on the activity of phosphodiesterase .
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Ascorbic Acid and splenic Cell c-(~
Vo1 .24, No . 17, 1979
Some characteristics of the process by which ascorbate influences cGMP metabolism, presumably through the activation of guanglate cyclase, were examined by testing the effect of agents with defined chemical actions to alter the ascorbate-induced cGMP response . The addition of EGTA (1 mM) to the Splenic cell suspensions, which resulted in a 15X lowering of the basal level of cGMP, did not impair the ability of ascorbate to increase cC~ . This is consistent with the Cam-independence of the effect of the oxidant periodate to elevate cGMP levels in these cells (5) and with the cationindependence of the ascorbate effect demonstrated in umbilical artery (4) . Aacorbate can participate in a non-enzymic, metal-catalyzed reaction with molecular oxygen which results is the generation of H20y (26) . Catalans (8 to 32 mg/ml of cell suspension), which would be expected to degrade any HpOp produced in the media, did not suppress the ascorbate-promoted increase in cellular cGMP accumulation . 1,2,4 Aminotriazole (0 .1, 1 .0 and 100 ml~ , which can enhance H20p-promoted events by inhibiting intracellular catalase act~.vity (27), also was without effect . The cyclo-oxygeaase pathway of fatty acid metabolism has been implicated in the ascorbate-promoted increase in cGMP in platelets (2) . However, preincubatioa of splenic cells (15 min, 37 ° ) with the cyclo-oxygenase inhibitors, aspirin (50 u~, indomethacin (50 ut~ or 5,8,11,14 eicosatetraynoic acid (50 uI~ (all of which lowered the basal cGMP concentration approximately 50X) had no significant effect on the magnitude of the increase in cGMP promoted by vitamin C . Diethyldithiocarbamic acid (DDC) (0 .05 and 0 .5 mM), a chelator with high affinity for transition metals and an effective free radical trapping agent (28), completely blocked the effect of ascorbate to elevate cGMP while lowering the basal concentrations 30-50X . The hindered phenolic anti-oxidants, 3t~utyl4-methyloxyphenol (BHA) and 4-methgl-2-6-di-t-butylphenol (BHT) also blocked the ascorbate-induced elevation in cGMP . These free radical scavengers had no effect on the basal cyclic nucleotide level . Discussion The relationship between ascorbic acid and splenic cell cGMP steadystate levels indicated by these results may have a two-fold significance . On one hand, it helps to further define a possible oxidative-reductive mechanism that may underly the cellular regulation of cG1+IP biosynthesis ; and, on the other, it identifies a pathway of splenic cell metabolism that may be regulated, at least in part, by vitamin C . That ascorbic acid acts as a physiological effector of cGt~ metabolism in these cells ie strongly suggested by the striking lowering (approximately 80X) of splenic cell cGMP concentration upon chronic vitamin C deprivation . Restoration of depleted levels of cGMP in isolated cells from scorbutic animals by concentrations of exogenous ascorbate that approximate the levels found in normal cells further implicates the vitamin as a possible natural cellular effector of cG[~IP metabolism is these cells . The Selectivity of vitamin C deprivation to affect the metabolism of this cyclic nucleotide in only splenic cells (and spleen) from among n~erous other cell types and tissues examined indicates that a relationship between vitamin C and cGMP metabolism of physiological significance may be restricted to only certain cell types . The effect of higher concentrations of exogenous ascorbic acid to promote cGl+lP accumulation ín umbilical artery (4), or in platelets (1,2,29) and macrophages (3) which showed no change in cGMP levels upon vitamin C deprivation, may sot be representative of the same relationship between
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vitamin C and cGMP uncovered in splenic celle . Ordinarily, ascorbic acid ie viewed as a substance which participates in redoz-related reactions because of its reducing potential, but its effect to elevate splenic cell c(~ paralleled the effect of oaidaata and was opposite to the effect of thíol reductaata . Thiol reductants also were effective in reversing the ascorbate-induced increase in cellular cGl~ . These results, combined with the recently reported evidence that guanylate cyclase from these cells can be activated by an apparent oxidative process (7), underscore the likelihood that the effects of ascorbate on splenic cell cGl~ metabolism result from the generation of oxidizing equivalents. Dehydroascorbíc acid can promote increases in Splenic cell c(~ steady-state levels sad activate the soluble and particulate forms of guanylate cyclase from these cells (7) . However, it is not likely that this metabolite represents the oaidiziag equivalent derived from ascorbate that affects cG~ metabolism in this system . Ten times more dehydroascorbate is required to elevate cG~iP levels in intact splenic cells sad to activate guanylate cyclase (7) in cell lysates than the concentrations of ascorbate effective in elevating cGMP levels in these cells . There was no detectable metabolism of dehydroascorbate to diketogulonic acid or to ascorbate in the intact or broken cell systems that would account for this greater requirement of dehydroascorbate . Furthermore, there ie no detectable conversion of ascorbate to dehydroascorbate during the time that as effect of the vitamin on cGMP metabolism in the intact cell is observable . Although the production of H20p can result upon interaction of ascorbate with ADP sad/or a transition metal (26), o~ddiziag equivalents in the form of Hp02 are not likely to be involved in the ascorbate-promoted elevation of cGt~ since catalane does not interfere with and 1,2,4 amisotriazole does not potentiate the effect ; also, HpOp (1-100 ~, in contrast to other oxidants (7,30), does not serve to elevate intact cell cGHP levels or to activate splenic cell guanylate cyclase. The results of the present studies also minimize the possibility of an involvement of Cam and the production of proataglaadine is the ascorbate effect on cGlíP metabolism in these cells. Schoepflin et al . (2) have reported that fatty acid cyclo-oaygenase inhibitors, auch as indomethacin, inhiHit ascorbate-induced cGi~ accumulation in platelets, but this was not found to be the case in the present studies nor with platelets in our earlier studies with ascorbate (1,29) . The basis for the disparate results are not known, but one difference besides the two different cell types employed is the two experiments was the use of the higher concentrations of ascorbate in the studies cited (2) . The transition metal requirement for the activation process, indicated by the inhibition by DDC, is similar to that previously shown for spontaneous activation of the soluble enzyme from lung (10) sad uterus (31) as well ae the activation of the enzyme from various sources by sodium azide (B) . The effectiveness of DDC as a free radical trapping agent ie, however, also recognized (28) and coupled with the observation that the ascorbate-induced elevation of splenic cell cGMP ie inhibited by the classical free radical scavengers, BHT and BHA, strongly suggests that free radical generation is involved in this action of ascorbate . In biological reactions is which ascorbate serves to promote oxidation which does not involve H20p production or conversion of the vitamin to its dehydro form (both of which seem unlikely is the present studies), it has bean postulated that the oaidiziag effector ie the labile, one electron oxidation product of ascorbate, moaodehydroascorbate (32-34) . Ascorbate oxidation, promoted either enzymically by ascorbate oaidase (35-37) or nonenzymically by comproportionation between ascorbate and
dehydroascorbate (38,39) or by transition metal catalysis (40) hen been shown to proceed according to the Michaelie formulation of a two-step oxidation which gives rise to a free radical intermediate (41) . The eaiatence of a
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Vol . 24, No . 17, 1979
semi-oxidized form of ascorbate which can serve as a relatively potent oxidant has been confirmed by electron spin resonance spectroscopy (37,42-46) . Guanylate cyclase activation by free radicals also has been suggested in reports on the activation of the enzyme by several nitroso-containing compounds (8, 15,17) . It also has been suggested that hydroxy-free radicals can activate guaaylate cyclase from several sources (18) . The direct evidence for a free radical mechanism of activation is not yet available, The demonstration that a relatively stable, more active form of the enzyme is measurable in lysates of intact cells treated with vitamin C indicates that the ascorbate-induced accumulation of cGMP in these cells results from an activation of guanylate cyclase . This is supported by the demonstration that ascorbate addition to the cell lysates also promotes enhanced guanylate cyclase activity although the undefined condition of preincubating the lysate (i .e ., before ascorbate addition) is required to maximize the effect . The effectiveness of the thiol reductiot, dithiothreitol, to convert the more active form of guanylate cyclase in lysates from ascorbate-treated cells to the less active basal state favors the view that ascorbate promotes guanylate cyclase activation in the intact cell by a process that involves oxidation of the enzyme or a closely associated regulatory component . These results are consistent with the concept that oxidation-reduction related processes represent one general mechanism by which cellular guanylate cyclase activity may be regulated and cellular steady-state levels of cGMP maintained or altered . Ackaawledgementa This research was supported by grants from the American Cancer Society (BC-166), the National Institutes of Health (NS-05979, NL-06314), and the Minnesota Leukemia Foundation . The preset address of Mari K . Haddox is Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724 . The present address of David B . Glass is Department of Pharmacology, School of Medicine, Emory University, Atlanta, Georgia 30322, Requests for reprints should be addressed to Dr . Nelson D . Goldberg, Department of Pharmacology, University of Minnesota, 105 Millard Hall, 435 Delaware Street, S .E ., Minneapolis, MN 55455 . Ref rences 1, 2, 3. 4. 5. 6.
7. 8.
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