Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 199-203 (1976) Naunyn-Schmiedeberg's

Archivesof Pharmacology �9 by Springer-Verlag 1976

Adrenal Cortex Adenylate Cyclase Is Ca 2 § Involved in A C T H St imulat ion?

H. Glossmann and H. Gips

Pharmakologisches Institut der Justus-Liebig-Universit/it, Frankfurter Strasse 107, D-6300 Giessen, Federal Republic of Germany

Summary. The chelator E G T A inhibits the activation of bovine adrenal cortex adenylate cyclase by ACTH. Hormonal response is restored by addition of Ca 2+, Mn 2 + and other cations which are able to significantly reduce the concentration of uncomplexed EGTA in the adenylate cyclase assay medium. Time course studies reveal that the enzyme in the activated state (induced by adenylate cyclase reagents and hormone or by pretreatment with hormone) is resistant to EGTA inhibition.

Key words." Adenylate cyclase - A C T H - EGTA.

I N T R O D U C T I O N

It has been generally accepted that calcium is involved in the action of A C T H on adenylate cyclase in adrenal cortex and adipose tissue (for review: Perkins, 1973). The evidence for the participation of the divalent cation in the activation of the enzyme by A C T H in broken cell preparations is indirect and rests on experi- ments with the chelator EGTA. EGTA abolishes effects of A C T H on adenylate cyclase from bovine adrenal cortex (B/Jr and Hechter, 1969a) and rat fat cell ghosts (Bfir and Hechter, 1969b; Birnbaumer and Rodbell, 1969). Since the inhibition by EGTA is overcome by addition of calcium (BS, r and Hechter, 1969a, b) a calcium requirement was postulated. We have performed experiments which indicate that alternative interpretations of the EGTA effects are possible.

M A T E R I A L S A N D METHODS 1

The sources for chemicals have been given in earlier reports (Gloss- mann and Gips, 1974, 1975). The preparation of partially purified

Send offprint requests to." H. Glossmann at the above address. 1 The abbreviations used are: cAMP, cyclic adenosine 3':5'- monophosphate; Gpp(NH)p, 5'-guanylyl-imidodiphosphate; EGTA, ethyleneglycol bis (/3-aminoethylether) N,N'-tetraacetic acid.

adrenal cortex plasma membranes has been described (Glossmann and Gips, 1975). Adenylate cyclase activity (ATP pyrophosphate- lyase, cyclizing, EC 4.6.1.1) was determined with [c~-32p]ATP as substrate and [32PlcAMP purified as described by Salomon et al. (1974). The reaction mixture contained 50 mM Tris-HC1 buffer pH 7.6, I mM cAMP, 2 mM 1-methyl-3-isobutylxanthine, 5 mM MgC12, 5 mM creatine phosphate, 0.6 mg/ml of creatine phospho- kinase, 1 mM dithiothreitol and the listed additions. [c~-32P]ATP was 0.1 or 0,25 raM. For fixed time point experiments the reaction volume was /50 ~tl. Time course studies were conducted with a reaction volume of 1.2 ml and 100 gl aliquots were removed at the given times. The reaction was initiated by addition of prewarmed (3 rain) enzyme solution and carried out at 30 ~ C. The concentration of membrane protein was 0.1 - 0.3 mg of protein per ml. Adenylate cyclase activity is expressed in nmoles cAMP per mg of protein. "Basal" is the activity in the absence of added stimulants.

Fixed time point experiments are always means from dupli- cates; time course experiments are single point determinations, The following final concentrations of additions were used: ACTHl_24 0.6gg/ml; Gpp(NH)p, 5 gM; GTP, 0.3p, M; NaF, 12 raM. EGTA was added as the Tris-salt.

RESULTS

EG TA Inhibition o f ACTH-Stimulated Adenylate Cyclase and Reversal by Ca 2+, Mn 2+ and Other Cations

Inclusion of EGTA in the adenylate cyclase assay medium leads a concentration dependent inhibition of the A C T H response as previously reported by others (Fig. 1). I f GTP (0.3 IxM) was added to the assay medium, adenylate cyclase activity (in the presence of ACTH) was enhanced (Glossmann and Gips, 1975). Under these conditions activity was particularly sensitive to EGTA inhibition and the chelator at low concentrations abolished nearly all effects of added GTP.

In more detailed experiments (not shown here) we found that 30 gM EGTA (which corresponds to about 40 nM uncomplexed chelator, if added Mg 2+ is considered the only competing cation in the adenyl-

200 H. Glossmann and H. Gips

3.0

A = ACTH+ G p p ( N H ) p B = Gpp(NH)p

ACTH " GTP C \ D : A C T H

2 . 5 \ E -- G T P

2.0

~ |

"~ 1.5

t 0 C

/

o 0.5 [- ,,.,,~_ - -= ; - o!

r >- o

O / i i t J 0 o.125 0.25 0.5 1.0

TOTAL EGTA [ m M ]

Fig. 1. Effects of EGTA on adenylate cyclase activity. ATP was 0.1 mM

ate cyclase assay mixture) was sufficient to reduce the effects of added GTP by more than 60 ~ .

Activities in the presence of Gpp(NH)p or GTP were enhanced by EGTA (Fig. 1); fluoride-stimulated activity (12 mM NaF) was not altered by the chelator (0 .1-1 raM) (not shown). Addition of Ca 2 + reversed the EGTA inhibition (Fig. 2b) and the initial value was reached at the equivalence point (0.2 mM in this experiment). If M n 2+ w a s added instead of Ca 2+, we obtained essentially the same result. In contrast to Ca 2 +, however, activity was enhanced if the concentration of M n 2+ exceeded that of the chelator. These results suggested that the effects of added cations (e.g. Ca 2+ or Mn 2+) below the equiv- alence point reflected their ability to complex free EGTA. We tested this with other cations (Table 1). C o 2+ , Sr 2+ and N i 2+ w e r e also able to reverse or partially reverse the inhibition of the ACTH response by the chelator. From the results in Fig. 2b and Table 1 it is evident that, with the notable exception of Mn 2 +, Ca 2+ and other cations inhibited adenylate cyclase activity in the absence of EGTA. The effects of added Ca 2+ on ACTH-stimulated adenylate cyclase were antagonized by added Mn 2 + and vice versa (Fig. 2a and Fig. 3).

M n §247

1.0 ~ AG'rH

Ca § 0.5 Ca"* : 0.2raM �9

Mn§ 0.2raM <~

a

0 ' ~ ' ' ' ' I I

'~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ACTH

< 0.

0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

C A T I O N C O N C E N T R A T I O N [ m M ]

Fig. 2. (a) (top) Competition between added Ca 2+ and Mn 2+ on ACTH-stimulated adenylate cyclase. The activity in the absence of added Ca 2+ or Mn 2+ is indicated by the dotted line. If Ca 2+ (0.2 raM) is present, adenylate cyclase activity is inhibited. Increasing concentrations of Mn z + reverse the inhibition and activity increases above the control value if Mn 2+ > Ca z+ (closed symbols). In the presence of Mn 2+ (0.2 mM) adenylate cyclase activity is enhanced. Increasing concentrations of Ca 2 + reverse the stimulation by Mn z + (open symbols). (b) (bottom) Reversal of EGTA inhibition by Mn 2+ or Ca 2+. The effects of adding either Ca 2+ or Mn z+ on adenylate cyclase activity [in the presence of ACTH and EGTA (0.2 raM)] are shown. ATP was 0.1 mM

Table 1. Effects of several cations on ACTH-stimulated adenylate cyclase in the absence and preser/ce of EGTA. Adenylate cyclase activity was measured as described under "Methods" in the presence of ACTH. ATP was 0.1 raM. EGTA, when added, was 0.25 raM. Membrane protein was 0.1 mg per ml. Mean values from triplicate assays. Basal activity was 0.19 nmoles cAMP/mg x 15 min

Cation added ' Adenylate cyclase activity (0.25 raM) (nmoles cAMP/rag x 15 min)

+ EGTA - EGTA

None 0.31 0.63 ZnC12 0.35 0 CoC12 0.51 0.44 SrC12 0.65 0.55 NiC12 0.45 0.42 CuC12 0.25 0 MnC12 0,65 0.95 FeCI 2 0.18 0.11 LaC13 0:38 0.03

Adrenal Cortex Adenylate Cyclase 201

Fig. 3 Effects of added Ca 2 + (left) and Mn 2 + (right) on adenylate cyclase activity. The reversal of Ca 2+ (0.42 raM) inhibition of adenylate cyclase (measured in the presence of ACTH and GTP) by increasing concentrations of Mn 2 + is shown on the right, ATP was 0.1 mM

.~ 0.8

0.7

0.6

~ o~

0.4

o.a

o

~ 5 1,0 1.5

4.0

3 .0

2.0

1.0

0 5

. ._ - -D

A C T H * G T P

+ G T P * C a +§

B,,a~ -------------~i

. . . . o'~ ,o , ;

Ca ++ added [rnM] Mn +~ added [mM]

Effects o f EGTA and Ca 2 + on the Time Course o f c A M P Production

The experiments shown above suggested that reversal of EGTA inhibition by Ca z § and other cations was due to the complexing of free EGTA in the enzyme reaction mixture. This could occur prior to the addition of enzyme and substrate and the experi- ments do not prove that inhibition of ACTH activa- tion by EGTA is truly reversible. In Fig. 4 a time course experiment is shown where EGTA or Ca 2§ were present at a concentration high enough to inhibit cAMP production to a similar degree. Addition of Ca 2 § to assays containing EGTA rapidly increased the rate and similar results (although different rates) were obtained by adding EGTA to assays containing Ca 2+. Addition of Ca 2§ to assays lacking EGTA lead to immediate inhibition of the enzyme, but addi- tion of EGTA (in the absence of added Ca 2 +) had no effect on the rate for at least 5 min (Fig. 4, Fig. 5). This was an unexpected finding. It suggested that EGTA effects were dependent on time and order of addition of the components of the adenylate cyclase assay mixture. If membranes were preincubated with hormone for 60 min at 0 ~ and subsequently added to an assay mixture containing EGTA, an increased rate of cAMP production persisted for 6 - 7 rain despite the presence of the chelator (Fig. 5).

DISCUSSION

Calcium ions are required for ACTH stimulation of lipolysis in adipose tissue (Katocs et al., 1974) and steroidogenesis in adrenaI cortex (Birmingham and Bartova, 1973). It was therefore an intriguing finding that EGTA inhibited ACTH activation of adenylate cyclase in broken cell preparations from both tissues.

|

1.o I~)

? ~

e. 0.5 -

o_

0 ' ~ I I I I i i I I i

5 I0 TIME [minutesl

Fig. 4. Effects of EGTA and Ca 2+ on the time course of cAMP production by bovine adrenal cortex membranes. ATP was 0.25 raM. In some experiments additions were made at 5.5 rain (arrow). A Time course in the presence of ACTH; B like in A bu( with 1 m M Ca 2 + and 1 mM EGTA; C EGTA (to 1 raM) was added to assays containing ACTH and Ca 2+ (1 mM); D Ca 2§ (to 1 raM) was added to assays containing ACTH; E Ca 2 § (to 1 raM) was added to assays containing ACTH and EGTA (1 mM); Fbasal activity (0.02 nmoles c A M P / m g x m i n ) ; G ACTH and EGTA (1 raM) were present (0.025 nmoles cAMP/rag x min); H ACTH and Ca 2 + (I raM) were present (0.024nmoles c A M P / m g x m i n ) ; for reasons of clarity the experimental points for curves G and H have been omitted from the figure, since they nearly coincide with those for F. The activities of adenylate cyclase are given in parentheses. I E G T A (to i raM) added to assay containing ACTH. The values obtained in this experiment are indicated by the closed symbols

This abolishment of hormonal response of adenylate cyclase by EGTA and reversal of inhibition by calcium is not unique for the ACTH-dependent

202 H. Glossmann and H. Gips

1.0 @(!~

J

c

n

0 . 5 <

co

O

0 i i i J l i I I i l i i 0 5 10

T I M E [ m i n u t e s ]

Fig. 5. Effects of preincubation of bovine adrenal cortex mem- branes with ACTH on EGTA effects. ATP was 0,25 mM. A Time course of cAMP production in the presence of ACTH; EGTA (to I mM) was added at 5.5 min; B membranes were preincubated for 60 rain at 0 ~ C with ACTH and after temperature equilibration added to the incubation medium containing EGTA (1 mM); C membranes were added to assays containing ACTH and EGTA (1 mM); D basal activity

enzyme: Lysine vasopressin activation of adenylate cyclase in porcine renal plasma membranes is inhibited by EGTA (Campbell et al., 1972) and EGTA inhibits the effects of oxytocin on the adenylate cyclase in frog bladder epithelia cells (Boeckart et al., 1972 ).

In the case of ACTH-stimulated adenylate cyclase Bfir and Hechter (1969a, b) concluded that traces of free Ca 2§ are necessary for hormonal activation. There is, however, no convincing evidence for a direct stimulatory action of added Ca 2 + (in the ab- sence of EGTA) on the ACTH response. Added Ca 2 § inhibits most adenylate cyclase systems and the adrenal cortex enzyme is no exception. The inhibitory action of Ca 2 + is not fully understood. We have found, e.g. that Mn 2+ can reverse the calcium inhibition. Steer and Levitzky (]975) recently reported similar findings for the turkey erythrocyte enzyme.

Our results show that EGTA inhibition of ACTH activation is not only reversed by addition of Ca 2 § but also by Mn 2§ Co 2+ or Sr 2§ Based on these observations alone it appears doubtful that free Ca 2 § participates in hormonal activation. Mn 2+ or other cations could be equal candidates.

Johnson and Sutherland (1973) found a similar reversal of EGTA inhibition for detergent-dispersed brain adenylate cyclase. They suggested that un-

complexed EGTA binds with high affinity to some metal tightly fixed in the adenylate cyclase complex. Complexing of free EGTA by addition of cations with high affinity for the chelator will shift the equi- librium and relieve the enzyme from inhibition. We would like to suggest that EGTA inhibition of hormonal activation in the adrenal cortex adenylate cyclase complex is mediated by a similar mechanism and is unrelated to the complexing of free Ca 2§ This hypothesis would open the question where EGTA binds and acts. Rodbell et al. (]974) have proposed a model for hormonal activation of adenylate cyclase in which hormones (via their receptors) facilitate the de-repression of a catalytic subunit by guanine nucleo- tides acting through regulatory sites z.

Experiments with labeled ACTH showed no effect of EGTA on high-affinity binding of the hormone to adrenal cortex membrane fractions (Lefkowitz et al., 1970). It is therefore conceivable that the chelator interferes with the coupling process but not with hormone-receptor interaction 3. GTP, in contrast to its analogues Gpp(NH)p and Gpp(CHz)p, has only intrinsic activity to activate bovine adrenal cortex adenylate cyclase when ACTH is present (Glossmann and Gips, 1975). The reason for this is unknown but could reflect an influence of the hormone on the turnover of GTP on regulatory nucleotide sites. Added GTP, as demonstrated above, is much less effective to enhance the ACTH response when EGTA at very low concentrations is present. EGTA, on the other hand, did not lead to an immediate inhibition of adenylate cyclase when added after the hormone. The enzyme was also rendered resistant to EGTA inhibition for several minutes if exposed to the hormone in the absence of adenylate cyclase reagents. This is not a surprising finding in view of recent kinetic data which demonstrated that the adenylate cyclase complex exists in different transi-

2 Hormonal activation of adenylate cyclase is often seen in the absence of added guanylnucleotides. It has been suggested that the regulatory site possesses an affinity for the substrate (Boeckart et al., 1972) or that the substrate is sufficiently contamined with guanine nucleotides (Rodbell et al., 1974). Londos and Rodbell (1975) observed that the enzyme preparation contained nucleotides which were removable by dialysis. We were unable to reduce the ACTH response of bovine adrenal cortex membranes by simple dialysis or washes. The possibility is still open that tightly bound nucleotides are present in the membrane fraction which can only be removed by chelation, activated charcoal or more drastic procedures. 3 In the multireceptor fat cell system the action of ACTH but not of epinephrine and glucagon is inhibited by the chelator. The effects of the latter hormones are even enhanced by EGTA (Birn- baumer and Rodbell, 1969). Among several alternatives, it could be considered that the primary events after hormone binding and the nueleotide requirements for ACTH and other hormones are different.

Adrenal Cortex Adenylate Cyclase 203

tion states and that the equilibrium between the differ- ent states can be a rather slow process (Rodbell et al., 1974).

As a working hypothesis we would like to propose that uncomplexed EGTA binds to the adenylate complex and thereby interferes with the ability of ACTH to activate adenylate cyclase through regu- latory nucleotide sites. It is, perhaps, not fortuitously that the affinity of the proposed binding site for EGTA must then be in the same order of magnitude as has been calculated for the free nucleotides acting inhibitory on the catalytic and stimulatory on the regulatory sites of the adenylate cyclase complex (Rodbell et al., 1974).

REFERENCES

Bfir, H. P., Hechter, O. : Adenyl cyclase and hormone action III: Calcium requirement for ACTH stimulation of adenyl cyclase. Biochem. biophys. Res. Commun. 35, 681- 686 (1969a)

B~ir, H. P., Hechter, O. : Adenyl cyclase and hormone action I: Effects of adrenocorticotropic hormone, glucagon, and epi- nephrine on the plasma membrane of rat fat cells. Proc. nat. Acad. Sci. (Wash.)63, 350-356 (1969b)

Birmingham, M. K., Bartova, A.: Effects of calcium and theo- phylline on ACTH- and dibutyryl cyclic AMP-stimulated steroidogenesis and glycolysis by intact mouse adrenal in vitro. Endocrinology 92, 743- 750 (1973)

Birnbaumer, L., Rodbell, M. : Adenyl cyclase in fat cells II hormone receptors. J. biol. Chem. 244, 3477-3482 (1969)

Boeckart, J., Roy, C., Jard, S. : Oxytocin-sensitive adenylate cyclase in frog bladder epithelia cells. Role of calcium, nucleotides, and other factors in hormonal stimulation. J. biol. Chem. 247, 7073 - 7081 (1972)

Campbell, B. J., Woodward, G., Borberg, V.: Calcium-mediated interactions between the antidiuretic hormone and renal plasma membranes. J. biol. Chem. 247, 6167-6175 (1972)

Glossmann, H., Gips, H. : Adrenal cortex adenylate cycIase: Com- parison between the action of GTP and 5'-guanylyl-imido- diphosphate on the particulate enzyme from bovine adrenal cortex and rat adrenals. Naunyn-Schmiedeberg's Arch. Pharma- col. 286, 239-294 (1974)

Glossmann, H., Gips, H. : Bovine adrenal cortex adenylate cyclase : Properties of the particulate enzyme and effects of guanyl nucleotides. Naunyn-Schmiedeberg's Arch. Pharmacol. 289, 77-97 (1975)

Johnson, R. A., Sutherland, E. W. : Detergent-dispersed adenylate cyclase from rat brain. Effects of fluoride, cations and chelators. J. biol. Chem. 248, 5114-5121 (1973)

Katocs, A. S., Jr., Largis, E. E., Allen, D. O.: Role of Ca 2 + in adrenocorticotropic hormone-stimulated lipolysis in the peri- fused fat ceil system. J. biol. Chem. 249, 2000-2004 (1974)

Lefkowitz, R. J., Roth, J., Pastan, I. : Effects of calcium on ACTH stimulated of the adrenal: Separation of hormone binding from adenyl cyclase activation. Nature (Lond.) 228, 864-866 (1970)

Londos, C., Rodbell, M. : Multiple inhibitory and activating effects of nucleotides and magnesium on adrenal adenylate cyclase. J. biol. Chem. 250, 3459-3465 (1975)

Perkins, J. P. : Adenyl cyclase. In: Greengard, P., Robinson, G. A. : Advances in cyclic nucleotide research III, pp. 1 - 64. New York: Raven Press 1973

Rodbell, M., Lin, M. C., Salomon, Y., Londos, C., Harwood, J. P., Martin, B. R., Rendell, M., Berman, M.: The role of adenine and guanine nucleotides in the activity and response of adenylate cyclase systems to hormones: evidence for multi-site transition states. Acta endocr. (Kbh.) Suppl. 191, 11 - 37 (1974)

Salomon, Y., Londos, C., Rodbell, M. : A highly sensitive adenylate assay. Analyt. Biochem. 58, 541-548 (1974)

Steer, M. L., Levitzky, A.: The control of adenylate cyclase by calcium in turkey erythrocyte ghosts. J. biol. Chem. 250, 2080- 2084 (1974)

Received July 19~Accepted October 21, 1975