Cyclic nucleotide metabolism during lymphocyte transformation

CELLULAR

43, 11-22 (1979)

IMMUNOLOGY

Cyclic

Nucleotide

I. Enzymatic

Lymphocyte

Mechanisms in Changes in CAMP and cGMP Concentration in Balb/c Mice BRUCE

Department

Metabolism during Transformation

of Biology,

J. SHENKER AND IRVING GRAY Georgetown Received

University. May

Washington,

D.C.

20057

22, 1978

Balb/c mouse spleen lymphocytes incubated from 0 to 30 min with the mitogen, lipopolysacchatide (LPS), were examined for alterations in concentration of cGMP and CAMP using radioimmunoassay. An optimal concentration of LPS, 10&lo6 cells/ml, caused an increase in the cGMP concentration which reached a maximum of 53% above control values 10 min after the addition of LPS. CAMP concentration also increased, showing two peaks, the first after 5 min to 32% above control values and the second after 30 min to 52% above control values. Although these changes in cyclic nucleotide concentration are small in comparison with other studies, they demonstrate that consistent and statistically significant data are obtained following transformation by a mitogen at its optimal concentration rather than at a concentration that causes maximum cyclic nucleotide changes. Enzymatic mechanisms were also investigated in order to explain the changes in cyclic nucleotide concentration during Balb/c mouse splenocyte transformation that were reported earlier. In cells incubated with LPS, the specific activity of adenylate cyclase increased more than twofold within 10min, while there was no change in guanyiate cyclase activity. Furthermore, cyclic nucleotide phosphodiesterase activity for both CAMP and cGMP increased by more than 20% over control values. These results explain the observed increase in CAMP, but not cGMP. It was demonstrated that CAMP was capable of inhibiting cGMP degradation by cyclic nucleotide phosphodiesterase by as much as 70%. The same is true for the effect of cGMP on CAMP degradation. LPS tended to inhibit the latter with no effect on the former. The relative affect was shown to be dependent on the cGMP/cAMPratio. Therefore, it is proposed that the elevation in cGMP concentration observed early in lymphocyte activation occurs as a consequence of the inhibition by each cyclic nucleotide on the hydrolysis of the other.

INTRODUCTION The addition of mitogen to a culture containing lymphocytes results in a complex series of events, initiated by the binding of mitogen to receptors on the cell surface and resulting in several biochemical and morphological changes (1,2). Mediation of the mitogenic signal from the plasma membrane to the interior of the cell is believed to be a function of the cyclic nucleotides (3). Presently there are two conflicting hypotheses regarding the role of cyclic nucleotides during lymphocyte transformation. The first, supported by Hadden and colleagues, proposes a negative role for CAMP and a positive role for cGMP (4); the second, supported by Parker and colleagues, proposes a positive role for CAMP (5). 11 0008-8749/79/03001l-12$02.00/O Copyright 0 1979by AcademicPress,Inc. All rights of reproductionin any form reserved.

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Analogs of CAMP as well as agents capable of elevating CAMP concentrations, such as theophylline, have been shown to inhibit lymphocyte activation (6-8), including inhibition of protein, RNA, and DNA synthesis. Elevated concentrations of CAMP have also been shown to inhibit several immune functions such as antibody production and cytolytic activity in mixed lymphocyte cultures (9-12). Furthermore, Warren et al. (13) have shown that cholera toxin (a stimulant of adenyl cyclase) elevates CAMP in vivo resulting in decreased graft rejection. Attempts to measure intracellular concentrations of CAMP directly have not provided clear results. Several investigations have shown early increases in CAMP within 20 min after the addition of mitogen (14, 15), while others have shown decreases within 10 min (16) and still others have been unable to detect any change (17, 18). Investigations using cGMP, dibutyryl cGMP, and 8-bromo-cGMP have shown that these agents are capable of stimulating DNA synthesis in lymphocytes (19). In fact, 8-Br-cGMP appears to be mitogenic for B cells (20). Furthermore, the addition of cGMP to lymphocyte cultures results in enhanced mitogen-induced activation of both B and T cells as well as the reversal of the inhibitory effects of CAMP (7,21, 22). Attempts to measure the intracellular concentrations of cGMP during lymphocyte activation have likewise provided mixed results. Several studies have shown several fold increases in cGMP within the first 20 min (4, 23, 24). Other investigators have been unable to detect any significant change in the intracellular concentrations of cGMP (16, 25). Cyclic nucleotides undoubtedly can alter immunologic responses. However, if they are indeed involved in lymphocyte activation, one should be able to detect changes in the metabolism of cyclic nucleotides early in the activation process. Unfortunately, there is considerable disagreement over the changes in cell content of both CAMP and cGMP during activation. The disparity of observations of reported results may be explained in several ways: (i) differences in cell populations used; (ii) differences in the processing of cell cultures for subsequent nucleotide measurements; and (iii) use of concentrations of mitogen that exceed that necessary for maximum stimulation. This last point is probably the most critical. Our studies have had a twofold purpose. First to ascertain the time course of the changes in both cyclic nucleotides and second to determine the enzymatic mechanisms responsible for the observed changes. MATERIALS

AND METHODS

Isolation of lymphocytes. Lymphocytes were isolated from the spleens of 6- to 12-week-old male Balb/c mice as previously reported (26, 27). Cell culture. Prior to the addition of cells, 12 x 75mm polystyrene snap cap culture tubes (Falcon 2054) containing culture medium, RPMI-1640 (Gibco), were preincubated at 37°C for at least 30 min when 0.1 ml containing 1 x lo6 lymphocytes was added, and incubated for 1 hr at 37°C before adding mitogen. To those cultures receiving lipopolysaccharide (LPS, E. cofi 055:B5, Difco, Detroit, Mich.), the mitogen was added in a volume of 0.1 ml of RPMI- 1640. The cultures were brought to a final volume of 1.0 ml with RPMI-1640 containing 10% heat-inactivated fetal calf serum (Gibco), 100units/ml of penicillin, and 100pg/ml of streptomycin (Gibco) and were incubated for 72 hr at 37°C in humidified air containing 5% CO,. Cell cultures for radioimmunoassay (RIA) were set up as described above with

CAMP AND cGMP CHANGES

IN CELL

TRANSFORMATION

13

the following changes. HBSS containing bicarbonate and Hepes buffer were substituted, volume for volume, for RPMI-1640. Fetal calf serum, penicillin, and streptomycin were omitted. Cultures were incubated under the same conditions for the time periods indicated. Cell cultures for the enzyme studies were prepared by adding 0.25 ml of a cell suspension (prepared as described above) containing 4 x 10’ cells/ml to each of several 12 x 75mm polystyrene culture tubes, yielding 1 x 10’ cells per culture. Mitogen was added, the volume was brought to 1.Oml with HBSS, and the culture was incubated as described above. Index of cellular transformation. The incorporation of tritium-labeled thymidine ([3H]TdR) into DNA was performed as previously described (28). The amount of [3H]TdR incorporated into 5% TCA insoluble material was determined by liquid scintillation counting and stimulation indexes were calculated by dividing the activity in LPS-treated cells by the activity in control cells. Stimulation Index (S.I.) =

dpm of [3H]TdR in cells with LPS dpm of [3H]TdR in cells without LPS

Radioimmunoassay of cyclic nucleotides. At specified times, cell cultures were removed from the incubator, agitated, and transferred with a Pasteur pipet to 13 x lOO-mmPyrex culture tubes. The cultures were rapidly frozen by placing the tubes in a beaker containing iso-pentane cooled with liquid nitrogen (approximate cooling rate of the cells was 200”C/min to a final temperature of about - 159’C). The frozen cultures were thawed in an ice bath for 15-30 min at which time 3 ml of ice-cold acetone were added and the suspension was allowed to stand for 15 min. Acetone suspensions were placed in a boiling water bath for 2-3 min and then were centrifuged for 15 min at 2800 rpm (11OOg) at 2-5°C. The supernatants were transferred to clean 13 x loo-mm Pyrex culture tubes and lyophilized. RIA for CAMP and cGMP was carried out under identical conditions as described by Steiner (29), using commercially available RIA kits (Schwarz/Mann, Orangeburg, N.Y.). Cross-reactivity of the RIA antibody with other nucleotides was determined by substituting various nucleotides for the samples. Lyophilized samples were resuspended in 0.5 ml of 0.05 M sodium acetate buffer (pH 6.2) and 0.3 ml was transferred to each of several 12 x 75-mm polypropylene culture tubes (Falcon 2036) maintained at 2-5°C. In addition to the sample, 0.1 ml of radioactive cyclic nucleotide (ScGMP or SCAMP-TME1251)and 0.1 ml of rabbit IgG anti-CAMP or anti-cGMP were added to the tubes. All RIA tubes contained a final volume of 0.5 ml. Standard curves covering the range of O.Ol- 10 pmol were made simultaneously with each RIA and assured maximum accuracy. In order to assess the efficiency of cyclic nucleotide recovery, 0.01 &i of 14C-labeled cyclic nucleotide was placed in some cultures at the time they were frozen. After lyophilization, these cultures were resuspended in 0.5 ml of distilled water and 0.1 ml was used to determine the total radioactivity recovered. Of the remaining solution, 0.02 ml was spotted on Whatman #l paper and chromatographed in ethanol: 10 N ammonium hydroxide:water (70: 10:20) by descending chromatography (30). The radioactive samples were cochromatographed with both internal and external standards located by using uv absorption.

14

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AND GRAY

Liquid scintillation counting. Radioactive samples were’ prepared as described above and transferred to plastic liquid scintillation vials (Poly X Vials, Beckman Instruments). Ten milliliters of scintillation fluid, Ready-Solv HP (Beckman Instruments), were added to the vials. Sample size varied with the analysis being carried out. The radioactivity was measured in a Beckman LS-1OOC liquid scintillation spectrometer to a + 1% counting error. Adenyfate cyclase assay. After lo-min incubation periods the cultures were frozen in iso-pentane cooled by liquid nitrogen. The frozen cultures were held at -20°C for 30 min and then placed in a 0°C ice bath for 15 min. The partially thawed cultures were transferred to and homogenized in a 2-ml Ten-Broeck homogenizer. The cyclase reaction was started by adding 0.1 ml of the homogenized cells to 0.4 ml of 40 mM Tris-HCl buffer, pH7.4, which was 4.0 mM in ATP, 5.0 mA4in MgS04, 24 mM in creatine phosphate, and contained 28 units creatine phosphokinase. The reaction was allowed to run for 10 min at 30°C when it was stopped by freezing. The amount of CAMP produced was determined by RIA as described. Guanyfate cyclase assay. Cultures containing 1 x 10’ cells were prepared, homogenized, and assayed as described for the adenyl cyclase assay substituting GTP for ATP and 3.3 mM MnSO, for the MgS04 in the reaction mixture. Cyclic nucleotide phosphodiesterase. Cultures containing 1 x 10’ cells were prepared and homogenized as described for the adenyl cyclase assay. After homogenization, 2-mercaptoethanol was added to the enzyme preparation (final concentration, 3.75 mM). The assay mixture contained 0.4 ml of 40 mM Tris-HCl, pH 7.4, containing 5.0 mM MgClz and 0.01 jKi [8-14C]cAMP (53 mCi/mmol, Schwarz/Mann) or [8-14C]cAMP (60 mCilmmo1, AmershamSearle). The reaction was started by adding 0.1 ml of the homogenate (enzyme preparation) to the assay mixture. The reaction was allowed to proceed for 10 min at 30°C and stopped by boiling for 2.5 min (31). Cobra venom (0.1 ml) (Ophiophagus hannah; 1 mg/ml in 40 mM Tris-HCl) was added to the assay mixture and incubated for 10 min at 30°C in order to convert the 5’ nucleotide to the corresponding nucleoside. This reaction was terminated by adding 1.Oml of a 1:3 slurry of Dowex 1 x 8 anion exchange resin in distilled water. The tubes were centrifuged and the supernatant containing the nucleoside was transferred to liquid scintillation vials and counted as previously described. The amount of protein in the crude cell homogenates was determined according to the method of Lowry et al. (32) using bovine serum albumin as a standard.

RESULTS Figure 1 shows that the maximum stimulation index 6.35 for lymphocytes from Balb/c mice occurs at a concentration of 10 pg of LPS/106 cells/ml. When radioactively labeled cyclic nucleotides were added to several cultures in order to assess the degree of nucleotide recovery during the extraction procedure, the results revealed that 93% of the [14C]cGMP was recovered. When cochromatographed, more than 99% of this activity remained with cGMP standards. Recovery of [14C]cAMP was found to be 95% of the total radioactivity and 91% of this activity chromatographed with CAMP standards. No crossreactivity of CAMP or GTP with cGMP occurred below lo+’ M or cGMP or CAMP below 10e7M or ATP for CAMP below 10v5M.

CAMP AND cGMP CHANGES

1 1

5

10 IS'S

IN CELL

TRANSFORMATION

20

50

25

CONCENTRATION

FIG. 1. LPS dose-response curve. The Stimulation experiments. each done in triplicate.

100

15

250

500

(&ml)

Indexes plotted represent the mean 2 SE of two

Changes in cGMP and CAMP of Balb/c mouse lymphocytes incubated with LPS are indicated in Fig. 2. There appears to be little increase in cGMP (solid line) until a peak at 10min; the increase to 153%of control values is statistically significant. The cGMP concentration has begun to return toward control values at 20 and 30 min. CAMP concentration appears to be biphasic: A peak occurring at 5 min (132% of control values) followed by a minimum at 10min and a second increase starting at 20 min (142% of control values) and still rising at 30 min (151%). Actual CAMP and cGMP levels for both control and experimental cell populations are shown in Table 1. Control cell CAMP values ranged from 0.27 to 0.38 pmol/106 cells, while CAMP concentration in activated cells increased to 0.53 pmol/106 cells at 5 min and returned to normal at 10min, but were again increased at 20 and 30 min, 0.49 and 0.73 pmol/106 cells, respectively. Control cell cGMP levels remained constant between 0.12 and 0.16 pmoY106cells while that in activated cells increased to 0.26 pmoY106 cells at 10 min. Figure 3 shows the cGMP/cAMP ratio plotted as a function of cell culture time. The cGMP/cAMP ratio of control cells remains relatively constant (0.47-0.39) between 1 and 20 min after an initial decrease. The fluctuations observed are primarily due to changes in the control level of CAMP. LPS-stimulated cells appear to have two elevations of the cGMP/cAMP ratio, the first at 2 min (0.58) and the second at 10 min (0.79). The second peak is due entirely to an increase in cGMP. However, the smaller, earlier peak derives from a combination of an increase in cGMP as well as a decrease in CAMP, but is not statistically significant. The cGMP/cAMP ratio shows two periods of decline; the first between 2 and 5 min to a value of 0.25 pmoY106cells and the second after 10 through 20 and 30 min (0.37 and 0.19 pmoU106cells, respectively). Both declines are due to significant increases in

SHENKER AND GRAY

INCUBATION TIME

(minutes)

FIG. 2. Changes in cellular cyclic nucleotide levels of Balb/c mouse lymphocytes. The cyclic nucleotide levels in the stimulated cells at each incubation period are shown as a percentage of the concentration in the control cell population (without LPS). The solid line represents changes in the cGMP and the broken line represents CAMP. The results shown are the mean ? SE of three experiments, each done in triplicate (the results of the 30 min incubation represents one triplicate experiment).

the cellular CAMP content. Results of the experiment to determine the activity of enzymes involved in cyclic nucleotide metabolism are shown in Table 2. The specific activity of adenylate cyclase from control cells was 0.28 pmol CAMP formed/l0 mm/50 pg of protein while that of transformed cells rose twofold to 0.58 pmol CAMP formed/l0 mm/50 pg protein. Guanylate cyclase from control and transformed cells showed no significant difference in activity (0.58 and 0.61 pmol cGMP formed/l0 mm/50 kg protein, respectively). Hydrolysis of CAMP by the TABLE 1 Cyclic Nucleotide Levels in “Normal”

Balb/c Mouse Lymphocytes CAMP (pmoVIOGcells)

cGMP (pmoYIOp cells) Incubation time (min) 0 1 2 5 10 20 30 a Mean + SE b Mean + SE * Significantly ** Significantly *** Significantly

w/o LPS 0.16 ? 0.15 2 0.15 * 0.16 + 0.14 2 0.15 f 0.12 2

0.02 0.01 0.02 0.01 0.02 0.02 O.Olb

WILPS 0.16 ? 0.16 f 0.17 ? 0.13 & 0.26 k 0.18 f 0.14 *

0.02 0.02 0.02 0.01 0.03* 0.02 O.Olb

of three experiments, each done in triplicate. of one experiment, in triplicate. different from control (P < 0.005). different from control (P < 0.05). different from control (P < 0.1).

WI0 LPS 0.27 0.32 0.34 0.30 0.33 0.38 0.43

2 2 2 2 + + t-

0.04 0.08 0.03 0.05 0.08 0.05 0.02*

WILPS 0.27 ? 0.04 0.27 k 0.06 0.25 ? 0.05 0.53 k 0.10** 0.33 Ifr 0.03 0.49 k 0.10*** 0.73 k 0.2W

CAMP AND cGMP CHANGES IN CELL TRANSFORMATION

i

s

16 INCUBATION

-h TIMC

17

xh

(minutes)

FIG. 3. cGMP/cAMP ratios of Balb/c mouse lymphocytes. The solid line represents cyclic nucleotide ratios of control cells (without LPS) and the broken line represents the ratios from experimental cells (with LPS).

phosphodiesterase in transformed cells increased by 20% over that observed in control cells. Similar results were observed for cGMP for which the rate of hydrolysis in transformed cells increased by 26% over control values. Assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase activities revealed a mechanism to account for the increase in cellular CAMP, that is, an increase in adenylate cyclase activity. However, because no change in activity was observed for guanylate cyclase, the explanation for increased cGMP concentrations remained obscure. Furthermore, the possibility of an interconversion of CAMP to cGMP was investigated to account for elevated cGMP levels. The results of these experiments revealed that no such mechanism was present. It was decided, therefore, to look at the effect each cyclic nucleotide had on the cyclic nucleotide phosphodiesterase. Figure 4 shows the effect of CAMP on the degradation of [‘4C]cGMP by cyclic nucleotide phosphodiesterase. Increasing the amount of CAMP resulted in greater suppression of cGMP hydrolysis to as little as 29% of the uninhibited value for control cells, and to 28% for LPS-stimulated cells. Statistical analysis of these data indicates no significant difference between the control and LPS-activated cell populations. Figure 5 shows that the maximum inhibition by cGMPof [r4C]cAMP hydrolysis in control cell homogenates was 48% while LPS appears to decrease the inhibition. A comparison with Fig. 4 shows that cGMP does not inhibit CAMP in control cells to the same extent that CAMP inhibits cGMP hydrolysis.

18

SHENKER AND GRAY TABLE 2 Activity of Enzymes Involved in Cyclic Nucleotide Metabolism Activity”.” (pmol or dpm/lO mm/50 pg protein) Enzyme Adenylate cyclase Guanylate cyclase Cyclic nucleotide Phosphodiesterase CAMP cGMP

w/o LPS

WlLPS

0.28 ? 0.1 pmol 0.58 + 0.2 pmol

0.58 f 0.05 pmol* 0.61 ? 0.2 pmol’

2269 f (2.04 x 2579 2 (2.34 x

2723 2 (2.48 x 3260 k (2.96 x

217 dpm lO-11mol) 88 dpm 10-r’ mol)

34 dpm** 10P1 mol) 102 dpm** IO-” mol)

n Mean -c SE of three experiments, each done in triplicate. b Cultures were incubated for IO min and then the cells were assayed for enzyme activity. c Not statistically different. * Significantly different from control (P < 0.001). ** Significantly different from control (P < 0.05).

DISCUSSION Several earlier studies have shown that mitogens alter cGMP levels in lymphocytes (4, 14- 16,23,24). In this study, optimal mitogenic concentrations of LPS were observed to elevate cGMP more than 50% above control values within 10 min after the addition of the mitogen. Although the increase in cGMP is not large in comparison to that observed by Hadden and co-workers (4), it does represent a consistent and statistically significant (P < 0.005) alteration in cGMP concentration. Furthermore, an increase in cGMP during the early stages of mitogenic stimulation correlates with the studies on the effect of cGMP on lymphocyte function (7, 9, 33). The CAMP measurements in these cells indicate that transformation at the optimal mitogenic concentration of LPS also has a profound effect on the cellular content of this cyclic nucleotide. Other investigations concerning mitogenic stimulation of lymphocytes and changes in CAMP concentration have provided a broad spectrum of results (14- 18). The data presented in this paper show that in lymphocytes incubated with LPS, there are two periods of increase in CAMP concentration. Elevations in CAMP first appear at 5 min and then again beginning at 20 min. While some previous work has shown that elevated CAMP concentrations tend to inhibit lymphocyte transformation (6-8), others have suggested that small increases in CAMP may be mitogenic to lymphocytes (33) and that small increases in this cyclic nucleotide are compatible with the early processes of lymphocyte transformation (5). Cook and co-workers (34) have shown that early elevations in CAMP can result in the stimulation of antibody synthesis in lymphocytes. There are several aspects of this study that should be considered when comparing these data with those from other similar studies. First, an optimal mitogenic concentration of LPS (10 pg/106 cells/ml) was used to stimulate the lymphocytes, as contrasted to a concentration used by others that yields maximum alteration in cyclic nucleotide metabolism. Second, very rapid freezing of cell cultures was

19

CAMP AND cGMP CHANGES IN CELL TRANSFORMATION

40--

20--

0 I 0

2

4

6

0

1

0.50

0.33

8 0.25

CAMP (x

10-l’ moles 1

cGMP”/cAMP

FIG. 4. Inhibition of [‘*C]cGMP hydrolysis in Balb/c mouse lymphocytes with changing CAMP concentrations. The enzyme assay was performed under conditions in which the amount of unlabeled CAMP was altered, but the amount of [YlcGMP remained constant. The solid circles represent the mean ? SE for lymphocytes cultured without LPS and the solid line represents the least squares regression line for the data. The open circles represent the mean ? SE for lymphocytes cultured with LPS and the broken line represents the least squares regression line of the data. Each data point is the product of three experiments, each done in triplicate. Cells for analysis were homogenized after IO min incubation.

r: 0

2

4

0

1

0.50

FIG. 5. Inhibition of [W]cAMP

6 0.33

8 0.25

cGNP (x

LO-lo moles)

cAMP”/cGMP

hydrolysis in Balb/c mouse lymphocytes with changing cGMP. The solid circles represent the mean -CSE for lymphocytes cultured without LPS and the solid line represents the least squares regression line of the data. The open circles represent the mean 2 SE for lymphocytes cultured with LPS and the broken line represents the least squares regression line of the data. Each data point is the product of three experiments, each done in triplicate. Cells for analysis were homogenized after 10 min incubation.

20

SHENKER

AND GRAY

employed to stop the incubations. Examination of Fig. 2 indicates that the changes in cyclic nucleotide concentrations are very abrupt and transient, particularly during the first 5 min. Thus, it is imperative that fast and efficient methods be used to arrest metabolic activity. Failure in this regard could result in a wide spectrum of results. Further, excessive steps in the purification of antigens for RIA can result in loss of the antigen at each step as well as the introduction of substances which might interfere with the RIA (35). The extraction of cyclic nucleotides in this study was essentially a one-step procedure involving the use of acetone. Acetone has the added property of high volatility, making it rather easy to remove from the samples. (Preliminary experiments performed in this investigation showed that it was extremely difficulut to remove TCA completely, thereby resulting in interference with the RIA.) These results from Balb/c mouse splenocytes provide consistent, as well as significant data demonstrating that transformation by a mitogen-in this case LPS-alters the cellular content of cyclic nucleotides (2). It should be emphasized that when the cellular content of CAMP and cGMP is measured information only on total cell content is obtained. It has been proposed that cyclic nucleotides are present in compartments within lymphocytes (2). If this is so, then it may also be that mitogens (or antigens) induce changes in cyclic nucleotide concentrations within a particular compartment. Therefore, the small change in total cyclic nucleotide concentration that is observed may, in fact, represent alterations of relatively greater magnitude within these compartments of the cell. Furthermore, it is conceivable that lymphocyte transformation is not directly dependent on the absolute concentration of the cyclic nucleotides, but rather on the ratio between the two. Thus, small changes in either or both nucleotides would result in a considerably large change in the ratio as shown in Fig. 3. In general, the cellular concentrations of cyclic nucleotides are determined through a delicate balance of synthetic and degradative metabolic pathways (36). The evidence obtained from studies on cyclic nucleotide synthesis is varied. Both adenylate cyclase and guanylate cyclase have been shown to be present in lymphocytes (19, 37). However, the influence of mitogens on adenylate cyclase appears to be inconsistent while they appear to have no effect on guanylate cyclase (2, 19). Degradation of cyclic nucleotides by cyclic nucleotide phosphodiesterase has been demonstrated in lymphocytes with relatively high activity associated with particulate cell fractions (19,38). However, there is a lack of information regarding changes in this enzyme(s) during lymphocyte activation by mitogens. Evidence from our study reveals that the specific activity of adneylate cyclase is elevated more than twofold (Table 2) in cells cultured with LPS. Thus accounting for elevations in CAMP presents no problem. Increases in cGMP, however, are not as simple to explain; no change in guanylate cyclase activity was observed in this investigation. Furthermore, no evidence for an interconversion of cyclic nucleotides was found although such a system has been previously described in frog heart tissue (39). Since elevated cGMP concentration did not appear to be due to changes in the synthetic pathway, it was decided to investigate the catabolic pathway associated with cGMP. The specific activity of cyclic nucleotide phosphodiesterase was determined with both CAMP and cGMP as substrates. This activity increased in the presence of LPS by more than 20% above control values for both CAMP and cGMP.

CAMP AND cGMP CHANGES

IN CELL

TRANSFORMATION

21

These results were perplexing when considering the changes in cellular cyclic nucleotide concentrations that were observed. Some type of competition between the cyclic nucleotides for cyclic nucleotide phosphodiesterase seemed to be a plausible mechanism to account for the changes observed in cGMP concentration. Inhibition studies were performed and the results indicate that both CAMP and cGMP are capable of altering the rate of degradation of the other. Similar results have been reported by others (38). The results of the inhibition experiments show that CAMP can inhibit the breakdown of cGMP by as much as 70%. It is also evident that LPS has no effect on this suppression. On the other hand, cGMP was shown to inhibit CAMP hydrolysis by as much as 60%, but, in the presence of LPS, inhibition by cGMP was depressed (Fig. 5). It would appear then, that during lymphocyte activation elevated CAMP concentration can be explained by increased adenylate cyclase activity, and the increase in cGMP can be accounted for as a consequence of the inhibition of the breakdown of cGMP by the elevated CAMP concentration. After 5 min of culture, the cGMP/cAMP ratio is reduced to 0.25. At this nucleotide ratio, approximately 65% inhibition of cGMP hydrolysis could be expected (Fig. 4). Therefore, it appears to be the inhibition by CAMP of cGMP hydrolysis that accounts for the subsequent elevation in cGMP. If such a mechanism is operating, one would expect a second rise in CAMP as cGMP returns to normal values because elevated cGMP concentrations would raise the cGMP/cAMP ratio, resulting in less hydrolysis of CAMP and more hydrolysis of cGMP. This second elevation in CAMP was observed and is probably due to both elevated adenylate cyclase activity and cGMP suppression of CAMP hydrolysis. Thus, an enzymatic mechanism is present to account for the alteration in cyclic nucleotide concentrations observed during lymphocyte activation. The mechanism involves alterations in synthetic and degradative pathways of both cyclic nucleotides. Several investigators have suggested that lymphocytes exist in a quiescent state (G,) but can be stimulated by mitogen and/or antigen (3,24). Such stimulation was hypothesized to be mediated by the cyclic nucleotides, which provide the signal to the lymphocytes so that they “awake” from the resting G, stage and enter the cell cycle (3, 40). It is evident from this study that the cellular concentrations of both CAMP and cGMP change early after the addition of mitogen. Therefore, one might conclude that both cyclic nucleotides play an important stimulatory role in lymphocyte activation. cGMP has been proposed to be the second messenger involved in the alteration of nuclear processes (24). Johnson and Hadden (41) have shown that within 10 min after the addition of mitogen, cGMP stimulated DNA-dependent RNA polymerase I activity. cGMP may also be involved in other altered nuclear processes. The end result then of the cyclic nucleotides activity is an altered cellular activity associated with the metabolic priming of the cells so that it can proceed through the process of transformation. Complete transformation most likely also depends on continued interaction of mitogen with the surface receptors (42). It has been demonstrated (25) that merely altering cellular levels of cyclic nucleotides is not sufficient for cell transformation. Thus it cannot be ruled out that the reported alterations in cyclic nucleotide concentrations may be the result of cellular activity not related to lymphocyte transformation such as mitogen-plasma membrane interactions or other secondary effects.

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