Adrenal cortex adenylate cyclase

Naunyn-Schmiedeberg's Arch. Pharmacol. 299, 175 - 185 (1977) Naunyn-Schmiedeberg's

Archives of Pharmacology �9 by Springer-Verlag 1977

Adrenal Cortex Adenylate Cyclase In vitro Modification of the Enzyme by Cholera Toxin

H. GLOSSMANN and C. J. STRUCK with technical assistance by C. KONRAD

Pharmakologisches Institut der Justus Liebig-Universitfit Giessen, Frankfurter Strasse 107, D-6300 Lahn-Giessen, Federal Republic of Germany

Summary. Pretreatment of rat adrenal particulate fractions with cholera toxin in vitro markedly changed the properties of the membrane-bound adenylate cyclase. The basal activity of the enzyme was increased after cholera toxin treatment. The main action of the toxin was on the Vma~ of the enzyme. In the absence of added GTP Lineweaver-Burk plots indicate a deviation from normal Michaelis-Menten kinetics with respect to substrate, the slopes being concave downward for control and toxin-treated membranes. Although hormonal stimulation of the enzyme was diminished in toxin-treated membranes, the hormone receptors were still functionally active as revealed after addition of Gpp(NH)p, GTP or GTPTS. The response to NaF was decreased in the toxin-treated membranes. Where- as GTP behaves as an antagonist (or a partial agonist with low intrinsic activity) compared to Gpp(NH)p in control membranes, it has similar intrinsic activity as Gpp(NH)p in the toxin-treated membranes. The results indicate that cholera toxin modification of the adenylate cyclase complex is located at the guanyl nucleotide sites or factors controlling the turnover of GTP at these sites. Cholera toxin modification may be a useful tool to investigate the role of guanyl nucleotide sites in the regulation of adenylate cyclase activity.

Key words: Cholera toxin - Guanyl nucleotides - Adenylate cyclase.

INTRODUCTION

We have recently reported that differences existed in the in vitro response of rat adrenal and bovine adrenal cortex adenylate cyclase with respect to ACTH and its analogue NPS-ACTH which is chemically modified at the tryptophan in position 9. The analogue com-

Send offprint requests to H. Glossmann at the above address

pared with ACTH is a partial agonist and has lower apparent affinity in the bovine adrenal cortex adenylate cyclase system but is an ACTH antagonist for rat adrenal adenylate cyclase. Upon addition of Gpp- (NH)p, NPS-ACTH behaved as a partial agonist for the rat adrenal enzyme (Glossmann and Struck, 1976).

The experiments suggested that Trp 9 in the ACTH molecule plays an important role by interacting with a region of the hormone receptor critical for the ex- pression of intrinsic activity. Serious doubts about the validity of these in vitro experiments with adenylate cyclase arised, however, since the analogue is an antagonist for the ACTH-mediated cAMP response in isolated rat adrenal cells (Moyle et al., 1973). In continuation of these studies we subsequently found1 that, although Gpp(NH)p could increase the intrinsic activity of the analogue in vitro, it could not increase its apparent affinity as was the case with the full agonist and other agonistic analogues (Glossmann and Struck, 1976). The ratio of affinities for ACTH and NPS- ACTH with Gpp(NH)p present was now close to that determined with isolated cells. This suggested that the adenylate cyclase system in the intact rat adrenal cell was operating in terms of affinity (but by no means in terms of intrinsic activity) as we found it in test tube experiments with Gpp(NH)p.

The question was still open why bovine adrenal cortex and rat adrenal adenylate cyclase responded differently to NPS-ACTH in the absence of Gpp(NH)p, The specifity of both ACTH receptors seemed to be very similar. There are a number of reasons why adenylate cyclase systems despite identical hormone recognition sites could respond differently to hormone analogues. The density of the receptors or their mobil- ity with respect to catalytic subunits could play a role. Differences could also exist in the coupling step

In collaboration with J. Ramachandran, UC San Francisco, U.S.A.

176 Naunyn-Schmiedeberg's Arch. Pharmacol. 299 (1977)

between receptors and the catalytic subuni ts in which guanyl nucleot ide sites and the factors control l ing their occupancy are critical. The key role of the guanyl nucleot ides was suggested by the f inding that rat adrenal adenylate cyclase obta ined the propert ies of bovine adrenal cortex adenylate cyclase by adding G p p ( N H ) p ( G l o s s m a n n and Struck, 1976).

We considered to modify the adrenal adenylate cyclase systems in vitro by chemical and other means. It is hoped that we may confer on m e m b r a n e - b o u n d adenylate cyclase new propert ies by selectively modify- ing receptors, guanyl nucleot ide sites and other factors par t ic ipat ing in the h o r m o n a l response. This could eventual ly enable us to unde r s t and why h o r m o n e analogues like N P S - A C T H can act as agonists or antagonis ts on different target tissues. The findings communica t ed in the present paper suggest that cholera toxin selectively at tacks the guanyl nucleot ide sites and factors cont ro l l ing the occupancy of these sites

in the adenylate cyclase complex. Cholera toxin may be a useful probe to unde r s t and the role of these com-

ponen ts in h o r m o n e action.

M A T E R I A L S A N D M E T H O D S 2

The sources for chemicals have been given in earlier reports (Gloss- mann and Struck, 1976). Myokinase (EC 2.7.4.3), and GTPTS were from Boehringer, Mannheim, Germany. ATP was from Sigma, Mtinchen. ACTH1 24 (Synacthen | was a gift from Ciba-Geigy AG., Basel, Switzerland.

Purification of ATP. The protocol for DEAE cellulose chromatography of ATP was followed as described by Kimura et al. (1976) except that [~32p] ATP and [3H]-GTP (1 x 106 dpm; 3 x 10 6 dpm, respectively) were added prior to chromatography. After one column run the ATP peak was collected as determined by optical density and [32p] radioactivity and rechromatographed on a 0.6 x 6 cm Dowex AG 50W-X8 (Biorad, Mtinchen) column as described by Clark and Seney (!976). The neutralized effluent was put over a 0.6 x 3 cm Chelex | 100 column (Biorad, Mfinchen) and kept frozen. The final contamination by GTP was estimated under the assump- tion that [3H] radioactivity in the collected ATP peak (corrected for spillover and [31p] decay) represented residual GTP. The estimate was, that we had removed 95 ~o of the GTP present in commercially available ATP.

Preparation of Rat Adrenal Particulate Fractions and Bovine Ad- renal Cortex Membranes. Rat adrenal particulate fractions were isolated as described elsewhere except that I mM dithiothreitol (DTT) was present in the medium (Glossmann and Gips, 1974). Sucrose gradient purified bovine adrenal cortex membranes were prepared as reported (Glossmann and Gips, 1975).

Pretreatment of Rat Particulate Fractions and Bovine Adrenal Mem- branes with Choleratoxin. The pretreatment consisted of three steps. In the first step cholera toxin was activated under conditions

2 The abbreviations used are: Gpp(NH)p, 5'-guanylylimidodiphosphate; GTPyS, guanosine-5-(~-thiotriphosphate); cyclic AMP, cAMP, adenosine-3',5'-monophosphate; EGTA, ethyleneglycolbis (fl-aminoethylether)-N, N'-tetraacetic acid; NPS-ACTH, 9-tryptophan (o-nitrophenylsulfenyl)-ACTH

slightly modified from those described by Gill (1976). In the second step freshly isolated particulate membrane preparations were incubated with activated toxin and cofactors. In the third step particulate membrane fractions were washed free from cofactors and un- bound toxin.

First step: Highly purified cholera toxin (Sattler et al., 1975) was preincubated with 0.02 ~ (w/v) sodium dodecyl sulfate, 1 mM dithiothreitol for 30 rain at 37 ~ C.

Second step: The freshly isolated particulate membrane fraction was divided into two equal aliquots (containing approximately 10 mg of protein) and incubated in a medium slightly modified from the conditions given by Wheeler et al. (1976). The medium contained: NAD, 1 mM; dithiothreitol, 2 mM; ATP (commercial), 5 mM; creatine-phosphate, 10 raM; MgC12, 5 raM; 100 mM Tris-HC1- buffer pH 7.4, creatinekinase i mg/ml. The preincubated toxin in dodecylsulfate and DTT was added to a final concentration of 125 or 250 p-g/ml. Controls received dodecylsulfate and DTT only. The final concentration of dodecylsulfate was 0.001 ~o (w/v). The incubation time was 20 rain at 30 ~ C.

Third step: After incubation toxin-treated membrans and controls were transferred into 40 ml ice-cold 20 mM NaHCO3, 1 mM dithiothreitol and spun for 15 min at 20000 x g (+2~176 The supernatants were decanted and the pellets after resuspension washed as above. Finally, the particulate preparations (controls and toxin-treated) were taken up in 4 ml 20 mM NaHCO3, 1 mM DTT, divided into aliquots and kept frozen in liquid nitrogen until use .

Adenylate Cyclase Assay. Except for the inclusion of myokinase (50 p-g/ml) the assay conditions were as has been described in detail (Glossmann and Struck, 1976). In brief: Prior to assay control and toxin-treated membranes are thawed and preincubated at I~ for 15 min with the additions (guanyl nucleotides, NaF, ACTH) indicated in the legends with NaN3 (5 mM) and MgClz (0.1 mM) present. The rest of the adenylate cyclase assay reagents was added and incubated for 15 min at 30~ Determinations of adenylate cyclase activity were done in triplicate. The concentration of [e32p]. ATP is indicated in the legends. It will be also stated if commercial or purified (see above) ATP was used. The final concentration of additions of GTP, Gpp(NH)p, GTPyS were 10 p-M; ACTH1-24 was 1 gM, if present, and NaF was 8 raM. For each experiment presented in the paper a different freshly prepared rat adrenal preparation (24-40 adrenals) has been employed. The values of adenylate cyclase activity for control preparations therefore show some variation as reported (Glossmann and Gips, 1974).

R E S U L T S

Effects o f Pre treatment on Adenylate Cyclase and Concentration-Response Curves f o r Cholera Toxin

In Table I an exper iment is shown, where a rat adrenal par t iculate p repara t ion was pretreated with cholera toxin (250 gg/ml) as described in detail unde r Materials and Methods. Contro ls received the same t rea tment except that cholera toxin was omit ted dur ing prein- cubat ion . After washing adenylate cyclase activity was de termined in the respective m e m b r a n e fractions with the addi t ions indicated in the legend. Since the quest ion arose dur ing pre l iminary experiments if the p re t rea tment with cofactors (and cholera toxin omitted) had a deleterious effect on the enzyme we chose a substrate concen t ra t ion of 0.1 m M to allow a direct compar i son with values publ ished earlier on rat adrenal

H. Glossmann and C. J. Struck: Cholera Toxin Modification of Adrenal Adenylate CycIase 177

Table 1. The effects of cholera toxin-treatment (250 gg/ml) on adenylate cyclase activity (in pmoles cAMP per mg of protein and 15 rain) in rat adrenal membranes in the absence of added stimulants, with GTP, Gpp(NH)p, NaF, ACTH present and combinations thereof. Both, purified and commercial ATP (0.105, 0.10 raM, respectively) have been used as substrates. Determinations were done in triplicate from the control and toxin-treated preparation. Results are mean values • S.D.

Substrate Addition Cholera toxin-treated Controls

none ACTH NaF none ACTH NaF

Purified ATP

Commercial ATP

none 75 • 13 89 • 2 3t0• 30 42• 5 72• 11 1058 • Gpp(NH)p 332 • 29 616 • 28 535 • 26 234 • 12 883 • 59 1024 • 54 GTP 236 • 9 497 • 17 517 • 43 86 • 16 166 • 15 1066 • 53

none 83 • 7 103 • 5 405 • 68 47 • 1 92 • 7 946 • 24 Gpp(NH)p 334 • 10 567 • 78 520 • 45 211 • 10 882 • 73 838 • 20 GTP 224• 16 453 • 488 • 55 • 4 158 • 3 937 •

adenylate cyclase not pretreated at all (Glossmann and Gips, 1974).

Our conclusion is that the pretreatment itself had no adverse effect on adenylate cyclase activity. It was found that inclusion of cholera toxin in the preincubation medium lead to an elevated basal activity, a decreased activity with NaF and an increased activity in the presence of GTP when hormone was absent. Com- parable results were obtained with purified and commercial ATP. We admit that membrane-bound guanyl nucleotides derived from commercial ATP (present during pretreatment) could still play a significant role. We noted, that GTP and Gpp(NH)p could increase adenylate cyclase activity in the presence of fluoride in the toxin-treated membranes. A similar finding has recently been reported for GTP when purified ATP was used as substrate for hepatic adenylate cyclase not pretreated with cholera toxin (Kimura et al., 1976). Neither GTP nor Gpp(NH)p had a pronounced effect on the activity in the presence of NaF when control membranes were tested. The same finding has been reported before for rat adrenal preparations not pretreated at all (Glossmann and Gips, 1974).

The results also suggested that adenylate cyclase activity determined in the presence of hormone was similar whether the membrane fraction received cholera toxin during the pretreatment period or not. This has been a consistent finding throughout this study.

The results presented in Table 1 were obtained with a rather high cholera toxin concentration. We decided to investigate the changes of adenylate cyclase with varying concentrations of toxin during pretreatment. Since there is evidence that the active subunit liberated from the toxin by detergent and dithiothreitol adsorbs to glass- and plasticware (Gill, 1976), we had to modify the experimental protocol by including a carrier protein to dilute the activated toxin prior to preincubation. The toxin was incubated with sodium-

dodecylsulfate and dithiothreitol as described and the solution adjusted to 10 mg/ml ovalbumin in 10 mM Tris-HC1, pH 7.4. Serial dilutions were performed in 10 mg/ml ovalbumin in Tris-HCl-buffer. The effects of cholera toxin on adenylate cyclase were concentration dependent. With the above protocol a nearly thousandfold dilution of cholera toxin was dem- onstrating significant effects on adenylate cyclase in particulate preparations exposed to it during preincubation. Cholera toxin at 0.17 gg/ml doubled basal activity (48 + 7.3, 98 _+ 2.8 pmoles cAMP formed per mg of protein and 15 min, respectively) and increased the activity in the presence of 10 gM GTP by a factor of three (77 _+ 4, 230 _+ 20 pmoles cAMP formed per mg of protein and 15 min, respectively). The complete concentration-response curve is shown in Figure 1. The figure illustrates that the effects of cholera toxin are maximal under the conditions used in all other experiments shown in this paper. They furthermore show that the changes we observe and dis- cuss in the properties of particulate adenylate cyclase at high concentrations of cholera toxin are (although not being maximal) also observed at much lower concentrations. We decided to use 125 gg/ml of cholera toxin for all further studies. This concentration seemed to be the optimal for the chosen preincubation conditions. In other experiments (not shown) toxin-pretreated membranes were washed more than twice. The observed changes of adenylate cyclase persisted. We conclude that a quasi covalent modification of the enzyme complex resulted from the pretreatment with toxin which cannot be reversed by simple washing procedures.

Substrate Affinity of Adenylate Cyclase and EGTA-Sensitivity of the ACTH Response

In order to determine whether cholera toxin treatment affects the interaction of substrate with adenytate


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Fig. 1. Dependence of adenylate cyclase activity on the concentration of cholera toxin during preincubation of rat adrenal particulate preparations. The experiment was started by activating the toxin with sodiumdodecylsulfate and dithiothreitol as described under Materials and Methods. The activated toxin was diluted with ovalbumin (10 rag/m1) as carrier protein. Samples received either ovalbumin or cholera toxin in ovalbumin. The final concentration of ovalbnmin in the preineubation mixture was 0.5 mg/ml, the cholera toxin concentration was as indicated in the figure. After preincubation the particulate preparations were washed by centrif- ugation, resuspended in 20 mM NaHCO3, I mM dithiothreitol and assayed for adenylate activity with the indicated additions. The ATP concentration in this experiment was 0.55 mM; purified ATP was employed. Mean values are shown _+ S.D. Note that the ability of Gpp(NH)p to stimulate adenylate cyclase activity in the presence of ACTH decreases as the ability of GTP increases to do so when the cholera toxin concentration during preincubation is increased

cyclase, enzyme activities were measured with and without GTP present and in the presence of GTP and ACTH. Both, commercial ATP and purified ATP were employed. The total Mg 2§ concentration of 5 mM was high enough to minimize levels of uncomplexed ATP. Lineweaver-Burk plots of the data show a deviation from normal Michaelis-Menten kinetics for both purified and commercial ATP with control and cholera toxin-treated membranes when GTP was absent (Fig. 2 a, b).

The main action of the toxin appears to be on the Vmax of the enzyme. There was a small increase in the Km value with respect to substrate for the toxin- treated enzyme. We do not attribute much significance to this finding since the Km value obtained for commercial ATP was also higher in the presence of GTP with control membranes.

We conclude that cholera toxin does not change the affinity of adenylate cyclase for Mg-ATP to any maj or extent. This is in complete accordance with data on rat liver adenylate cyclase modified by cholera toxin (Franks, 1976).

It has been mentioned above that adenylate cyclase activity was similar in the presence of hormone whether the membrane fraction was exposed to cholera toxin during pretreatment or not. Since it could not be excluded that the hormone receptors or events close to it were adversely affected by pretreatment with toxin, it was tested if the hormonal response of adenylate cyclase was still EGTA-sensitive. As reported elsewhere (Glossmann and Gips, 1976), the inhibition of the ACTH response by EGTA is only clearly seen when the chelator is added prior or with ACTH. The results are shown in Figure 3. We conclude that the ACTH response of the toxin-treated adenylate cyclase preparation retained its EGTA sensitivity.

It can be also seen from the figure that to the extent the effect of GTP on adenylate cyclase was facilitated by hormone, it was inhibited by the chelator irrespec- tive if the guanyl nucleotide itself showed little (as in control membranes) or marked activation (as in cholera toxin-treated membranes) of adenylate cyclase.

Effects of the Guanyl Nucleotides

Our data strongly suggested that the pretreatment with cholera toxin changed the properties of adenylate cyctase with respect to Gpp(NH)p and GTP.

A concentration-response experiment for both nucleotides is shown in Table 2. In the absence of hormone GTP is much less effective than Gpp(NH)p to increase adenylate cyclase activity in control membranes. This was not the case in cholera toxin-treated membranes. The maximum activity of adenylate cyclase in the presence of hormone and Gpp(NH)p

H. Glossmann and C. J. Struck: Cholera Toxin Modification of Adrenal Adenylate Cyclase 179

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Fig. 2a. Dependence of adenylate cyclase activity on ATP concentration (purified and commercial ATP). Controis (open symbols) and cholera toxin-treated (125 btg/ml) membranes (closed symbols) were prepared as described in Materials and Methods. Activity of adenylate cyclase was determined in triplicates (mean values are shown, for reasons of clarity only, standard deviations are omitted except for the data obtained with ACTH plus GTP added). Activity in the absence of added stimulants : O <>: with GTP added : zX /x ; with GTP plus ACTH added : D- []

Fig. 2 b Reciprocal plots showing the data of Figure 2a obtained with purified ATP. Note that the data for basal activity are concave downward for controls and somewhat less for cholera toxin-treated membranes; no K,, value could be calculated for these data. The following K,, (mM) and Vm,x (nmoles per mg of protein and 15 min) values have been calculated by least-square fit computer analysis of the transformed data (control membranes) : GTP: 0.128, 0.114; GTP plus ACTH: 0.116, 0.485. Cholera toxin-treated membranes: GTP: 0.133, 0.571. GTP plus ACTH: 0.142, 1.428 (data with purified ATP). The following results were obtained with commercial ATP. Control membranes: GTP: 0.118, 0.154; GTP plus ACTH: 0.146, 0.497. Cholera toxin-treated membranes: GTP: 0.160, 0.641 ; GTP plus ACTH: 0.154, 1.490. The meaning of the symbols is as given in Figure 2a

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When both nucleotides, GTP and Gpp(NH)p, were added simultaneously (Fig. 4) GTP could inhibit

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An interesting aspect of the experiments depicted in Table 1 and 2 is, that neither GTP, nor Gpp(NH)p with hormone present could increase adenylate cyclase activity to the same extent in the toxin-treated


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Fig. 3. Effects of EGTA on the activity of rat adrenal particulate adenylate cyclase. Control and cholera toxin-treated membranes were prepared as described above and adenylate cyclase activity determined in the absence or presence of 1 mM EGTA (as Tris-salt). Stippled columns: EGTA present; open columns: no EGTA added. Means from triplicate determinations _+ S.D. Purified ATP (0.25 mM) was used as substrate

Table 2. Effects of GTP and Gpp(NH)p at varying concentrations on adenylate cyclase activity (in pmoles cAMP per mg of protein and 15 min) in the presence or absence of ACTH in control and cholera-toxin-pretreated membranes. Purified ATP (0.25 mM) was used as substrate. Cholera Toxin was 125 gg/ml during preincubation

Addition [gM] Cholera toxin-treated Controls

- ACTH + ACTH ~ - ACTH 4- ACTH

Gpp(NH)p

GTP

0 133 • 13 230 • 14 46 • 6 219 • 16 0.03 176 • 10 296 • 14 74 • 2 437 • 17 0.10 249 • 23 408 • 14 125 • 14 803 • 14 0.30 358 • 11 625 • 40 224 • 24 1299 • 14 1.00 461 • 24 853 • 280 • 10 1748 • 105

10.00 542 • 11 1297 • 53 330 • 11 2196 • 72

0 t33 • 13 230 • 14 46 • 6 219 • 16 0.03 184• 10 341 • 19 56 • 9 326 • 26 0.10 225 • 3 507 • 37 52• 6 434• 66 0.30 276 • 12 716 • 52 61 • 4 532 • 2 1.00 344 • 21 998 • 63 95 • 17 529 • 11

10.00 356 • 16 1048 • 29 79 • 15 617 • 15

p repara t ions as seen with G p p ( N H ) p and A C T H in

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m o n e present the max imal activities o f both enzyme prepara t ions were similar (Table 3). It was also no ted

that in the absence o f A C T H and with G T P ? S present,

adenyla te cyclase act ivi ty was always h igher in the

tox in- t rea ted prepara t ion . C o m b i n a t i o n s o f the guanyl

nucleot ides were also tested. G T P in equ imola r con- cent ra t ions appears to be m o r e effective to inhibit

the effects o f G p p ( N H ) p than those o f GTPTS. The

c o m b i n a t i o n o f G p p ( N H ) p and GTPTS leads tO an adenyla te cyclase activity which is similar to the one observed for each nucleot ide separately. In o ther

exper iments ITP (not shown) at 10 g M was only


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Table 3. Effects of GTP7S alone and in combination with GTP or Gpp(NH)p on adenylate cyclase activity in the presence or absence of ACTH. Membranes were pretreated with cholera toxin (125 gg/ml) as described, controls were pretreated similarly except that cholera toxin was omitted. Otherwise the incubation conditions were as in Table 2. Results are expressed in pmoles cAMP per mg of protein and 15 min. Purified ATP (0.25 mM) was used as substrate

Additions Controls Cholera toxin-treated

-ACTH +ACTH --ACTH +ACTH

none 75_+ 8 355 -- 15 188 +_ 9 325 • 9 Gpp(NH)p 320 • 15 2114 _+ 108 537 _+ 23 1440 • 14 GTP 77_+ 13 876_+ 45 410_+ 13 1239 • 56 GTP,S 341 _+ 16 2213 _+ 181 723 _+ 29 1896 _+ 186 Gpp(NH)p + GTP 147 + 32 1267 _+ 33 445 _+ 36 1421 • 48 Gpp(NH)p + GTPTS 346 _+ 26 2250 _+ 80 709 _+ 30 2021 _+ 53 GTP + GTPyS 252 • 23 1888 • 248 586 4- 13 1863 • 120

slightly less effective than G T P in toxin- t reated membranes in the absence of A C T H . We conclude that cholera toxin t rea tment increases the abil i ty of G T P to activate adrenal adenylate cyclase in the absence of A C T H and decreases the abil i ty of G p p ( N H ) p to exert its s t imula tory effects in the presence of hormone . Since the responses of adenylate cyclase to guanyl nucleot ides are often non- l inea r with respect to t ime our results ob ta ined with fixed incuba t ion times have to be confirmed and extended by t ime-course studies.

Experiments with Bovine Adrenal Cortex Adenylate Cyclase

In order to see whether the changes of rat adrenal

adenylate cyclase after cholera toxin t rea tment could be observed with other adenylate cyclase systems we have per formed experiments with a sucrose gradient purified bovine adrenal cortex p lasma m e m b r a n e frac- t ion. Since we have no t yet established the relat ion- ship between cholera toxin concen t ra t ion and adeny-


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Fig.5. Effects of cholera toxin-pretreatment on adenylate cyclase activity in bovine adrenal cortex membranes. A bovine adrenal cortex plasma membrane fraction was pretreated with cholera toxin (125 gg/ml) as described in Materials and Methods. Controls were pretreated exactly under the same conditions except that cholera toxin was omitted. Stippled columns: cholera toxin-pretreated membranes; open columns : controls. Determinations were performed in triplicate. Purified ATP (0.25 mM) was used as substrate

late cyclase modification we cannot generalize our findings. However, so far similar changes after toxin pretreatment were seen as with the rat adrenal adenylate cyclase system. An example is presented in Figure 5. Basal activity increased after toxin treatment, the fluoride response was diminished and with ACTH added adenylate cyclase activity was similar whether toxin was present during preincubation or not. As seen with rat adrenal adenylate cyclase, GTPTS was more active in the absence of hormone in toxin- treated membranes than in control membranes. GTP was ineffective to increase adenylate cyclase activity in the absence of hormone in control membranes but was clearly effective in the toxin-treated membranes.

In the presence of Gpp(NH)p and ACTH adenylate cyclase activity was slightly higher in toxin-treated membranes compared with controls in marked contrast to the rat adrenal adenylate cyclase system. At the present time we can offer no explanation for the difference but as mentioned in the introduction both enzymes display a different behaviour with respect to ACTH analogues and guanyl nucleotides. Gpp(NH)p induces a more than 15-fold increase in the apparent affinity of ACTH for the rat adrenal enzyme but does

not sh{ft the affinity for the bovine adrenal cortex adenylate cyclase system (Glossmann and Struck, 1976). We may speculate that the targets for cholera toxin are identical in both adenylate cyclase systems but that membranes may differ in the state, avail- ability, density and occupancy of the targets.

DISCUSSION

The results reported in the above paper show that pretreatment of adrenal particulate preparations with cholera toxin in the presence of various cofactors leads to a persistent change in the properties of adenylate cyclase. Although we present no experimental evidence for a cofactor (e.g. NAD) requirement and cannot completely rule out that a contaminant and not cholera toxin is responsible for the observed changes there is reason to believe that this may be not the case.

The increase in basal activity of adenylate cyclase after cholera toxin treatment has been reported by many others (Beckman et al., 1974; Flores and Sharp, 1975; Franks, 1976; Flores et al., 1976; Gill, 1976). The decrease in the response to fluoride ion which we found


has also been shown before (Sharp and Hynie, 1971; Kantor, 1975; Berkenbile and Delaney, 1976). A re- duction of the hormone effect after cholera toxin treatment has been recently reported (Berkenbile and Delaney, 1976). Nevertheless, a word of caution must be added. A diminished response of adenylate cyclase to sodium fluoride was seen after exposure of membrane preparations to adenylate cyclase assay reagents only (Rodbell, 1975). Adenylate cyclase preparations are sometimes poorly activated by hormones. We have recently found that this was the case with a crude guinea-pig myocardial particulate preparation. Upon preincubation at 37 ~ C (in the absence of adenylate cyclase assay reagents) the basal activity markedly decreased but isoprenaline or Gpp(NH)p were more effective with respect to basal activity than before (Krawietz et al., 1976). These examples are given to emphasize that certain changes which we could attribute to the action of cholera toxin on adenylate cyclase may be elicited by manipulation of membrane preparations in vitro without toxin being added. ATP and other components of the adenylate cyclase assay mixture could have played a significant but still poorly defined role in the changes reported above. Since structural information about the com- position of the adenylate cyclase system is absent or far from being complete we will interprete our observations within the framework of a widely accepted model (Rodbell et al., 1975) namely, that hormones via their receptors activate adenylate cyclase through guanyl nucleotide sites. Since GTP is often much less effective than certain GTP analogues like Gpp(NH)p, Gpp(CHa)p or GTPyS (Pfeuffer and Helmreich, :1975), additional controlling factors had to be postulated, e.g. enzymes which hydrolysed GTP at or close to its site or transferred its terminal phosphate to an un- known acceptor. A GTP'ase activity with high affinity for GTP has been demonstrated in the turkey erythrocyte membrane by Cassel and Selinger (1976). They have however not reported what the effects of GTPTS and Gpp(NH)p were on the GTP'ase. The above model does not account for hormonal stimulation in the absence of guanyl nucleotides. The argument was, that either commercial ATP was sufficiently contamin- ated by guanyl nucleotides or that alternatively ATP has lower but still significant affinity to the regulatory nucleotide sites. Experiments with highly purified ATP (Kimura et al., 1976), demonstrated that hormones can activate adenylate cyclase in the absence of added GTP. These experiments could not, on the other hand, exclude the possibility that enzyme source contained trace amounts of bound guanyl nucleotides and that ATP was needed, e.g. for a transphosphorylation step. Our experiments show that purified ATP even at much lower concentrations as employed by Kimura

et al. (1976) allows the hormone to increase the number of active adenylate cyclase systems 3. In the absence of added GTP and hormone Lineweaver-Burk analysis revealed a marked deviation from normal Michaelis- Menten kinetics with respect t o MgATP. This is in complete accordance with the results by Londos and Rodbell (1975). There are many alternative interpretations for the deviation; the most likely one is substrate activation (Garbers and Johnson, 1975). The experiments with purified ATP were started when we found that adenylate cyclase in cholera toxin- treated membranes was markedly more responsive to GTP in the absence of added hormone compared with control membranes. The differences which we observed with the two preparations of substrate are too small to allow firm conclusions on the role of GTP contamination under the experimental conditions used here.

The main feature of the toxin-treated enzyme preparation was the increased effectiveness of GTP to convert adenylate cyclase systems to the active state in the absence of added hormone. In this respect the toxin treatment had similarity to the action of hormone on adenylate cyclase activity in control membranes. Inhibition of bulk hydrolysis of GTP in the medium cannot be the main action of the toxin. We have previously reported that bulk [?P32]-GTP hydrolysis in the medium was inhibited by increasing the concentration of creatine phosphate or ATP. Under both conditions a GTP effect on adenylate cyclase was not detectable or diminished (Glossmann and Gips, 1975). Thus, hydrolysis of GTP in the medium and at or close to the G-sites are independent processes. GTP is an antagonist with respect to Gpp(NH)p when a fixed incubation time (e.g. 10 or 15 min) is used and both nucleotides are added simultaneously (Glossmann and Gips, 1974, 1975). We have shown that GTP like Gpp(NH)p could increase the rate of cyclic AMP formation by adrenal adenylate cyclase during the first two or three minutes of incubation, (Glossmann and Gips, 1974). Thereafter, the rate of cyclic AMP production in contrast to experiments with Gpp(NH)p rapidly declined. The rate was then indistinguishable from an adenylate cyclase preparation not being exposed to GTP. There was, however, a remarkable difference between the two conditions: The enzyme having been exposed to GTP was resistant to an equimolar concentration of Gpp(NH)p added later; the rate increased only very slowly in contrast to control preparations. Conversely, if Gpp(NH)p was

3 For the sake of simplicity we follow the proposal by Hammes and Rodbell (1976) and Maguire et al. (1975) that adenylate cyclase systems may exist in active and inactive states. The activity observed could then (with certain limitations) reflect the proportion of the total adenyiate cyclase systems in active state


added first, G T P could only very slowly decrease the rate of cyclic A M P product ion (Glossmann and Gips, 1974). It appears, that the nucleotide which had prior access to the guanyl nucleotide sites determined, for the times observed, the fraction of adenylate cyclase systems in active state. I f both nucleotides are added simultaneously, the nucleotide which was in excess would determine the effect as predicted by theory (Ariens, 1964). Cholera toxin treatment increased the intrinsic activity of GTP compared with Gpp(NH)p . We demonstrated this not only by measuring the activity of adenylate cyclase in the presence of each nucleotide added separately but also by adding them simultaneously: G T P could only to a very limited extent inhibit the effects of G p p ( N H ) p on adenylate cyclase, in marked contrast to experiments with control membranes or membranes not pretreated at all (Glossmann and Gips, 1974). There is one plausible interpretation for this observation: The toxin-treatment inhibited the rapid conversion of G T P on or close to the sites where it acts in the adenylate cyclase system.

Franks (1976) investigated the kinetic properties of a cholera toxin-modified liver cell m e m b r a n e adenylate cyclase. He found, that the effect of G T P on control and toxin-treated adenylate cyclase was identical and suggested that G T P and cholera toxin are noninteracting. This is in contrast to our findings and interpretations with adrenal adenylate cyclase. Of interest are the effects of G p p ( N H ) p and GTPyS. The former nucleotide cannot liberate a terminal phosphate; the latter can donate a thiophosphate (Eckstein, 1975). In control membranes G p p ( N H ) p could convert a certain fraction of adenylate cyclase systems into the active state in the absence of hormone. G T P was much less effective in this respect. In the presence of hormone, GTPyS and G p p ( N H ) p were almost equally potent to fully convert the adenylate cyclase systems to the active state in control membranes. In toxin-treated membranes G T P (especially at low concentrations, Table 2) was nearly equally effective as G p p ( N H ) p but still much less than GTP~S. GTPTS was, however, always more effective than Gpp- (NH)p in the absence of hormone in toxin-treated membranes compared with control membranes. We offer the following interpretation for the observed changes: To the extent the adenylate cyclase systems are modified by the toxin, the hormone is unable to convert them into the active state if the guanyl nucleotide sites are occupied by G p p ( N H ) p ; the reverse may be true for GTP. GTPyS may have some additive effect to the action of the toxin and may act on both modified or unmodified adenylate cyclase systems. Flores and Sharp (1975) have concluded f rom their studies that cholera toxin and the G T P analogue Gpp- (NH)p act on the same regulatory mechanism.

Our studies extend these observations and give support to the hypothesis that cholera toxin modifies a component in the adenylate cyclase system which regulates G T P turnover. We have used cholera toxin to prepare a modified adrenal adenylate cyclase system which, unlike the native enzyme, is highly responsive to the naturally occurring guanyl nucleotide GTP. Our findings could help to understand the activation of adenylate cyclase by cholera toxin in intact cells as well as to clarify the role of guanyl nucleotides in hormonal activation.

Acknowledgements. We would like to thank Dr. Staerk, Behring- werke, Marburg/Lahn for a gift of highly purified cholera toxin and Dr. Sattler, Institut f/it Physiologische Chemic, Marburg/ Lahn for valuable suggestions. Deutsche Forschungsgemeinschaft supported us with a grant (GI 48/10).

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Received May 13/Accepted July 1, 1977

NOTE ADDED IN PROOF

Two papers appeared after submission of the manuscript which may help to clarify the role of contaminating guanyl nucleotides (membrane-bound and substrate-derived) in the increased basal activity seen after cholera toxin treatment. Levinson and Blume (J. Biol. Chem. 252, 3766-3774, 1977) report that the basal activity of neuroblastoma adenylate cyclase (pretreated in situ with cholera toxin) was only increased with App(NH)p as substrate when a regenerating system was added. They suggest, that membrane- bound guanyl nucleotides were responsible for the increased basal activity of the toxin-treated enzyme.

Kimura and Nagata (J. Biol. Chem. 252, 3829-3835, 1977) have now purified commercial ATP by three successive column chromatographies on DEAE cellulose including a 7 M urea step (we have used only one DEAE cellulose step). Kimura and Nagata now conclude that GTP is absolutely required for the glucagon- stimulated adenylate cyclase in liver cell membranes when this highly purified ATP is used as substrate.

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