Naunyn-Schmiedeberg's Arch. Pharmacol. 294, 199-206 (1976) Naunyn-Schmiedeberg's

Archives of Pharmacology �9 by Springer-Verlag 1976

Adrenal Cortex Adenylate Cyclase In v i t ro Ac t iv i ty o f A C T H F r a g m e n t s a n d A n a l o g u e s

H. GLOSSMANN and C. J. STRUCK

Pharmakologisches Institut der Justus Liebig-Universitfit, Frankfurter Strasse 107, D-6300 Giegen

S u m m a r y . The ability of ACTH fragments and of an ACTH analogue [9-tryptophan(o-nitrophenylsul- fenyl)] corticotropin-(1 - 24)-tetracosapeptide[Trp- (Nps) 9 ACTH1-24] to stimulate adenylate cyclase in bovine adrenal cortex membranes and a crude mem- brane fraction from rat adrenals has been determined.

Partial agonists like Trp (Nps) 9 ACTH1 24 dis- played intrinsic activity in the rat adrenal prepara- tion only if tested in the presence of 5'-guanylylimi- dodiphosphate [Gpp(NH)p]. On the other hand, no addition of Gpp(NH)p was necessary to demonstrate intrinsic activity of Trp(Nps) 9 ACTH1-24 for bovine adrenal cortex adenylate cyclase. A large decrease (15-fold) of the apparent Km values for ACTHI-a4, ACTH1-23 and ACTH~_ ~7 was observed with the rat adrenal preparation when Gpp(NH)p was added. The shift in apparent Km values for ACTHl-24 and ACTHa-23 for the bovine adrenal cortex adenylate cyclase system was small or insignificant when Gpp- (NH)p was added.

The observations suggest that the hormone re- ceptor facilitates the action of guanylnucleotide sites in the membrane. When guanylnucleotide sites are occupied by Gpp(NH)p even weak interactions of the hormone receptor with e.g. partial agonists are propagated to the catalytic subunits of the adenylate cyclase complex resulting in enhanced activity. The differences in adenylate cyclase activation with hor- mone fragments or analogues and different target tissues may rather reflect the state of the coupling process involving guanylnucleotide binding sites of the isolated membrane fraction than differences in the receptor itself.

K e y w o r d s : ACTH - NPS-ACTH - Adenylate cy- clase.

Send offprint requests to: H. Glossmann at the above address.

INTRODUCTION

According to a widely accepted model (Rodbell et al., 1974, 1975) three functional sites are identifiable in the adenylate cyclase complex: A hormone receptor, a nucleotide binding site with specificity for guanyl- nucleotides and a catalytic site. The nucleotide binding sites play the key role in the hormonal activation process of the enzyme. Certain GTP analogues like Gpp(NH)p can even render activation by hormone agonists irreversible (Schramm and Rodbell, 1975) and activated adenylate cyclase is eluted with ease by detergents from membranes pretreated with hor- mones and Gpp(NH)p (Pfeuffer and Helmreich, 1975; Glossmann, 1975). In bovine adrenal cortex mem- branes guanylnucleotide binding sites interact with the angiotensin receptor (Glossmann et al., 1974) and modify adenylate cyclase activation by ACTH (Glossmann and Gips, 1975). In preliminary experi- ments we observed that a rat adrenal preparation did not demonstrate activation of adenylate cyclase by certain ACTH fragments and analogues whereas the bovine adrenal cortex enzyme did. It is reported below that these differences between rat and bovine adrenal cortex adenylate cyclase are more apparent than real and may not reflect intrinsic differences of the receptor but rather differences in the coupling process.

MATERIALS AND METHODS 1

The sources for chemicals have been given in earlier reports (Gloss- mann and Gips, 1974, 1975). 2-nitro-phenylsulfenyl-chloride was from Fluka AG, Buchs, Switzerland. Trp(Nps) 9 ACTHl-24 was prepared as described by Ramachandran and Lee (1970a) and purified by ascending paper chromatography (Whatman No. 1,

1 The abbreviations used are." Gpp(NH)p, 5'-guanylyl imidodi- phosphate; cyclic AMP, cyclic adenosine 3',5'-monophosphate; Trp(Nps) 9 ACTH1-24, [9-tryptophan (o-nitrophenylsulfenyl)]corti- cotropin-(l - 24)-tetracosapeptide.

200 Naunyn-Schmiedeberg's Arch. Pharmacot. 294 (i976)

butanol: acetic acid: pyridine: wltter, 30 : 6: 24: 20) according to Seelig and Sayers (1973). The modified peptide was eluted with 10 ~ acetic acid and lyophilized. The following peptides (references for synthesis are given and the abbreviations used in the text are in parentheses) were from Hoechst AG, Frankfurt, Germany: [//-Ala 1, Lys 17] ACTH-(I - 17)-heptadekapeptide-4 amino-n-butylamide acetate, (ACTHl-17), Geiger (1971); corticotropin-(7-10)-tetra- peptide (ACTHT-10), Hofmann and Lande (1961); Glutaroyl- [Lys 17] ACTH-(6-17)-dodekapeptide-4-amino-n-butylamide ac- etate, (ACTH6 17), Geiger and Sandow (in preparation); [/?-AI~0] ACTH-(1-9)-nonapeptide-8-amino-n-octyl acetate, (ACTH~-9), Geiger and Sandow (in preparation); ACTH-(l-23)-tricosapep- tide-amid acetate, (ACTH~ -z3); Geiger et al. (1964); [Lys ~7] ACTH- (11 - 17)-heptapeptide-4-amino-n-butylamide, (ACTH11 - 1 v), Geiger (1971). The following peptides were from Ciba-Geigy AG, Basel, Switzerland : Corticotropin-(11 - 24)-tetradecapeptide, (ACTHn -24); corticotropin-(1 - 10)-decapeptide (ACTH1 -s0); cor- ticotropin-(1-24)-tetracosapeptide (ACTHI-2,0, Kappeler and Schwyzer (1963).

Stock solutions of the peptides were prepared at a concen- tration of 1 mg/ml in 0.01 N HC1 using siliconized glass ware. Serial dilutions of the peptides were freshly prepared for each experi- ment, The peptide diluent contained bovine serum albumin (5 rag/ ml) and 0.5 mg/ml Trasylot | (a gift from Bayer AG, Leverkusen, Germany) in 10 mM TRIS-HCI, pH 7.4. The preparation of bovine adrenal cortex membranes and of rat adrenal particulate fraction has.been described (Glossmann and Gips, 1974).

Adenylate Cyclase Assay. For evaluation of concentration-response curves, a three-step incubation was performed: In the first step membranes were incubated with dithiothreitol in the absence or presence of Gpp(NH)p (10 min) at pH 7.4 (10 mM TRIS-HC1). During the second step (10 rain) membranes were exposed to peptide diluent or hormones in diluent. In the last step adenylate cyclase reagents were added and the test tubes incubated for 15 rain. The first two incubation periods were performed at the temperature of melting ice, the last incubation at 30 ~ C. The final concentrations of reagents were: albumin, 0.4mg/mi; Trasylol | 0.04mg/ml; MgC12, 5 mM; creatine phosphate, 5 mM; cyclic AMP, 1 mM; isobutyl-methylxanthine, 2 raM; creatine phosphokinase, 0.6rag/ ml; dithiothreitol, I raM; TRIS-HCl-buffer, 55 raM; Gpp(NH)p 5 gM, if present; [e 32p]-ATP, 0.25 mM (60-120 cpm/pmole). The final pH was 7.6. The reaction was terminated and sap cyclic AMP isolated as described by Salomon et al. (1974). The final concentration of membrane protein was kept between 0.3 and 0.9 mg/ml; the final reaction volume was 60 ~tl. Duplicates agreed within 7 ~ or better.

Since some of the peptides were in limited supply agonistic activity of the peptides was screened for in rat and bovine mem- branes. Subsequently a narrow concentration range was tested with both enzyme preparations in the absence or presence of Gpp(NH)p. Each experiment was repeated twice with different membrane preparations; representative examples are shown. Probit analysis of concentration-response curves was performed as de- scribed by Weber (1961).

R E S U L T S

The Bovine Adrenal Cortex Adenylate Cyclase System

In ear l ier exper imen t s it was no ted tha t h o r m o n a l s t imula t ion o f adeny la t e cyclase f rom bovine adrena l cor tex was a t ime-dependen t process . A t 0.75 n M A C T H a 24 up to 4 min were requ i red to ob t a in a signif icant enhancemen t o f enzymat ic ac t iv i ty above

2.4

2.1

1.8

1.5

_~ 1.2

0.9

0.6

(,9 0.3

0 0

Fig. 1.

c) o --[E!N

I I I I I i I i I 10-1 10 o 101 10 ~ 10 3 10 4 -105 106

PEPTIDE CONCENTRATION [ng/ml ]

Action of ACTH fragments on bovine adrenal cortex adenylate cyclase in the absence of Gpp(NH)p. The fragments of ACTH in this and other figures are identified by an abbreviation (subscript only). Conditions were as described under "Materials and Methods"

con t ro l s ( G l o s s m a n n and Gips , 1975). The t ime- dependen t ac t iva t ion even at sa tu ra t ing concen t ra - t ions o f h o r m o n e was m o r e p r o n o u n c e d with the ra t ad rena l m e m b r a n e p r e p a r a t i o n when G p p ( N H ) p was presen t ( G l o s s m a n n and Gips , 1974). Cond i t i ons were the re fore chosen which a l lowed s t imulants to come near equ i l ib r ium with the i r respect ive sites p r io r to the a d d i t i o n o f adeny la te cyclase reagents . A tota l p r e inc uba t i on t ime o f the m e m b r a n e f rac t ion o f 20 min was found to be sufficient.

Whe re a s ha l f -max ima l ac t iva t ion o f bovine ad- renal cor tex adeny la te cyclase occur red at a b o u t 60 ng /ml o f A C T H 1 - 2 4 when the h o r m o n e was a d d e d toge ther wi th adeny la t e cyclase reagents ( G l o s s m a n n and Gips , 1975), ha l f ma x ima l ac t iva t ion af ter prein- c u b a t i o n o f m e m b r a n e s wi th h o r m o n e was now ob- served at 4 ng /ml (Fig. 1, Tab le 1). The f ragment A C T H I - z 3 p r o d u c e d essent ial ly the same max ima l response as A C T H l - 2 4 and A C T H I - 1 7 behaved l ike a pa r t i a l agon is t wi th reduced affinity.

In the ra t ad rena l p r e p a r a t i o n (see below) inhibi- t ion was seen at high concen t ra t ions o f A C T H I - 1 7 and the concen t r a t i on - r e sponse curve was bell shaped. The f r agmen t A C T H 6 - 1 v had also intr insic act ivi ty to s t imula te bovine ad rena l cor tex adeny la te cyclase. F r a g m e n t s A C T H t - 9 and A C T H ~ - l o were active, a l t hough a t concen t r a t ions several o rders o f magni - t ude h igher c o m p a r e d wi th A C T H I - z e and

A C T H I - z B . The f ragments ACTHl l -2 ,~ , A C T H H - l v and

ACTHT-~o were inact ive in concen t ra t ions up to

H. Glossmann and C. J. Struck: In vitro Activity of ACTH Fragments 201

Table 1. Apparent Kra values (concentration of peptide in ng/ml which increased adenylate cyclase activity to 50 % of the maximal response seen with the full agonist ACTH~ 24) for different peptide fragments. The Km values were estimated from probit analysis of concentration- response curves. The mean value from two independent determinations (values in parentheses) is given. In cases where no Km value could be determined (n.d.) but the peptide had reproducible and significant intrinsic activity this is indicated by (+ ) = at 50 or 100 lag fragment/ml less than 10 %; + = 10 -20%, + + = more than 20 % of the activation seen with ACTIrti-24 (at 1 - 1 0 lag/ml). The biological activities of ACTH1-2a, ACTHI-17, ACTHT-lo, ACTH,-9 were (rat): 100 I.E./rag, 800 I.E./mg, 0, less than 1 I.E./mg0 respectively. (Geiger and Sandow, private communication). The dimension of the Km value [ng/ml) does not account for the different molecular weights of the peptide fragments. Since apparent Km values of the fragments are compared between the two adenyiate cyclase preparations (with and without Gpp(NH)p] the dimension will suffice

Rat adrenal membranes Bovine adrenal cortex membranes

Peptide no Gpp(NH)p with Gpp(NH)p no Gpp(NH)p with Gpp(NH)p

1 - 2 4 90 (80, 100) 5.0 (4,6) 1 - 2 3 100 (110, 90) 5.5 (6.5) 1 - 1 7 5000 (6000, 4000) 320 (300, 340) 6 - 1 7 n.d. ( + ) n.d. + + 1 - 9 -- n . d . + 1 - 1 0 - n . d . +

11-24 -- ( + ) 11 -17 - -- 7 - 1 0 - -

4.0 (3.5, 4.5) 1.8 (1.8, 1.8) 25 (30, 20) 25 (25, 25)

1000 (1200, 800) 110 (90, 120) 1 0 6 ~ 1 0 5

n.d. + n.d. + n.d. + n.d. +

0 .8

0.5 c

E LO

x 0.4

E \

0.3 E c

13_ 0.2

J CO0.] (D

0 4 1 I 0 100

o

o

i I I I I I

10 ~ 10 2 103 10'* 105 1 0 6

PEPTIDE CONCENTRATION [ng/ml]

0.7

0.6

- - 0 . 5 ._=

• 0,4

0.3

0.2

0,1 (..3

7

J /

41 I i I I i I I I

10 -1 10 ~ 101 10 2 10 a 10 4 10 b 10 6

PEPTIDE CONCENTRATION [ n g / r n l ]

Fig. 2. (a) (left) : Action of ACTH fragments on rat adrenal adenylate cyclase. The concentration-response curve for ACTHl_23 has been omitted for reasons of clarity. The peptide ACTH1-9 (not shown) was inactive as were ACTH1 -lo, ACTHv-lo, ACTHu -iv and ACTHtt 24. (b) (right) : Same as in (a), but with Gpp(NH)p present during preincubation

100 gg/ml (Fig. 1). When concentration-response curves were performed with membranes preincubated with Gpp(NH)p, none of the peptides which failed to stimulate adenylate cyclase in the absence of the GTP analogue had intrinsic activity in its presence. In particular, neither ACTHil-24 nor ACTHll- lv or ACTH7_ 10 gained intrinsic activity but ACTH1-17 was converted to a full agonist. Preincubation with Gpp(NH)p reduced the Km value for ACTH1-24 by a factor of two (Table 1). The Km value for ACTH1-23

was left unchanged, but those for ACTHl-17 and for ACTH6 17 were reduced by a factor of about ten.

The Rat Adrenal Adenylate Cyclase System

In the absence of Gpp(NH)p, ACTH1-24 and ACTHl-23 (Fig. 2a, ACTH1-23 not shown for rea- sons of clarity) had similar intrinsic activities to stimu- late rat adrenal cyclase. Their Km values were found to be very similar (90 ng/ml versus 100 ng/ml).

202 Naunyn-Schmiedeberg's Arch. Pharmacol. 294 (1976)

I I I I I

5 �9 I - -

o cr a- 4

3

i I i I i 0 1 2 3 4 LOGlo PEPTIDE CONCENTRATION[ng/ml]

Fig. 3. Probit analysis of the activation of rat adrenal adenylate cyclase by ACTHl-17 and ACTHI-z4 in the absence or presence of Gpp(NH)p. 1 ACTH1-24 [with Gpp(NH)p]; 2 ACTHI-z~ [no Gpp(NH)p; 3 ACTH~-17 [with Gpp(NH)p]; 4 ACTHl-17 [no Gpp(NH)p]

ACTHI-~v stimulated rat adrenal adenylate cyclase and inhibition occurred at high concentrations of the hormone fragment. ACTH6-17 enhanced rat adrenal adenylate cyclase to a very small extent; the other hormone fragments were essentially inactive in con- centrations up to 100 gg/ml. Large shifts in the Km values were observed when Gpp(NH)p was present during the first incubation period (Table 1, Fig. 3). Moreover, hormone fragments which were essentially inactive in the absence of the GTP analogue, like ACTHI - lo and possibly ACTH11 - 24 gained intrinsic activity (Fig. 2b). The peptide fragment ACTH~-~7 had nearly the same intrinsic activity as ACTH1-24, but its inhibitory action at higher concentrations was also more pronounced when Gpp(NH)p was present (Fig. 2b).

Experiments with a Modified ACTH1- z4

It is evident that the experiments shown above lack an important control. In no case the specificity of adenylate cyclase activation by fragments of the natural hormone has been proven by using a specific antagonist. The o-nitrophenyl sulphenyl derivatives of adrenocorticotrophin in which the tryptophan residue of the hormone is modified have been useful tools in the evaluation of ACTH interaction with its receptors in target tissues. Trp(Nps) 9 ACTH is a potent inhibitor of the lipolytic action of ACTH on rat fat cells (Ramachandran and Lee, 1970a) and does not stimulate rat fat cell ghost adenylate cyclase (Rama- chandran and Lee, 1970b). The derivative has very little potency to stimulate cyclic AMP production

in isolated adrenal cells but stimulates steroid synthesis to nearly the same extent as ACTH does, although with much less potency (Moyle et al., 1973). In ad- dition, it appeared to inhibit the effects of the un- modified hormone on cyclic AMP production but not on steroid synthesis (Moyle et al., 1973). We prepared Trp(Nps) 9 ACTHl-24 and tested its ac- tivity on bovine and rat adrenal adenylate cyclase.

The derivative had approximately 50~ of the intrinsic activity compared with unmodified ACTH1-24 in the bovine adrenal cortex adenylate cyclase system and its apparent affinity was at least one order of magnitude lower. In the presence of Gpp(NH)p the intrinsic activity of Trp(Nps) 9 ACTH1-24 was similar to that of ACTH1-24 (results not shown).

The rat adrenal adenylate cyclase system dem- onstrated only very small increases upon addition of Trp(Nps) 9 ACTH1-24. The maximum stimulation achieved with the derivative was approximately 3 of that seen with ACTH1-24 (Fig. 4a).

In the presence of Gpp(NH)p, Trp(Nps) 9 ACTH1-2~ gained intrinsic activity and reached 25 of that seen with ACTH1 24. If the derivative is in- deed a partial agonist it should fulfill several criteria: A combination of serial concentrations of ACTH1-24 combined with a constant concentration of the deri- vative should result in a parallel shift of the concen- tration response curves above the intersection point. The intersection point interpolated from different concentration-response curves represents the situa- tion where the response is independent of the con- centration of the derivative. On the other hand, serial concentrations of Trp(Nps) 9 ACTH1-2e tested in the presence of a constant concentration of the full agonist should lead to the same maximal effect (de- termined by the intrinsic activity of the partial ago- nist)-independent of the effect produced by the full agonist seen in the absence of the derivative. As can be seen from Figure 4b and Figure 5 this indeed proved to be the case. It was concluded from these findings that Trp(Nps) 9 ACTH1 -24 is a partial agonist for both adenylate cyclase systems (although apparent- ly only in the presence of Gpp(NH)p with the rat adrenal preparation) and not particularly useful as specific antagonist.

DISCUSSION

The structure-activity requirements of the ACTH receptor in isolated adrenal cells for the functional response (steroid production) and cyclic AMP in- crease have been evaluated by Seelig et al. 0975). One of the major findings in this study was, that the

H. Glossmann and C. J. Struck: In vitro Activity of ACTH Fragments 203

Fig. 4. (a) (left) : Action ofTrp(Nps) 9 loo ACTHI-z4 and ACTH1-24 on rat adrenal adenylate cyclase. The results 90- are expressed in percent of the maximal stimulation seen with Bo- ACTH1-24 and Gpp(NH)p. The incubation conditions in this 70- experiment and in Figure 5 were different from those given under 60- "Materials and Methods": The preincubation steps were omitted

O 5 0 - and prewarmed membrane fraction directly added to the otherwise •

4 0 - complete adenylate cyclase assay system (ATP = 0.1 mM) contain-

I;::: 3 0 - ing hormones and additions [e.g. > Gpp(NH)p]. [closed symbols : "~ Gpp(NH)ppresent; open symbols : , > , zo- Gpp(NH)p absent]. (b) (right): Response of rat adrenal lo- adenylate cyclase to Trp(Nps) 9 ACTH1-24 in the absence (closed symbols) or presence of different concentrations of ACTH1 _ 24 (open symbols). Gpp(NH)p was present during incubation at a final concentration of 5 gM

O" 0 10 .9

ACTH1 24 I# plus Gpp(NH);

[ 5'u ~x, t

I NPS-AOTH ' - 24 ~ lus

10 -8 10 -7 10 -6 1~-5 10-4

Pept ide concentrat ion[molar ]

100 90 1 Gpp(NH)p : 5~JM

o - 4 1 , , ,

10 .7 10 -6 10 -5

NPS -ACTH 1 24 [molar]

0,5-

0.4

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75 225

750 2250

T NPS - ACTH

1-24 [ nanomolar]

J / i i i i

0 10 .9 10 -8 10 .7 10 -6 10 .5

ACTH 1 24 (Molar) Fig. 5. Response of rat adrenal adenylate cyclase to ACTH1 24 in the absence (closed symbols) or presence (open symbols) of Trp(Nps) 9 ACTH1-24. Gpp(NH)p was present during incubation at a final concentration of 5 gM

structural requirements for maximal cyclic A M P produc t ion appear to differ f rom those for maximal steroidogenesis. The increase in cellular cyclic A M P , according to a dual receptor model (cyclic A M P not

obl igatory intermediate), may not be directly related to the functional response. On the other hand, a single receptor site-reserve model (cyclic A M P obl igatory intermediate) could also explain the apparent discrep- ancies between cyclic A M P increases and the ob- served rate o f cort icosterone product ion. The latter theory predicts that a partial agonist for cyclic A M P produc t ion can still be a full agonist for the func- tional response.

The A C T H receptor can also be characterized in vitro by its activity to enhance the activity o f mem- b rane-bound adenylate cyclase in broken target cell preparat ions. It follows f rom above that a corre- lation between functional response and in vitro activity (adenylate cyclase stimulation) o f A C T H fragments and analogues is o f limited value.

We at tempted to compare the in vitro activity o f some A C T H fragments and analogues for two dif- ferent target tissues. Very similar to results obtained by Jard et al. (1975) with vasopressin-sensitive pig kidney adenylate cyclase, a pre incubat ion step in- creased the sensitivity o f adrenal adenylate cyclase towards low concentra t ions o f hormone . It is likely but not proven for our experiments that the prein- cubat ion step was necessary to achieve equilibrium between the h o r m o n e and its receptors. Bovine adrenal cortex adrenal cyclase had high affinity for ACTH1 -24. The apparent Km (4 ng/ml = 1.2 nM) was close to the value found with isolated bovine adrenal cells (4 n M ; Peyt reman et al., /973). Under identical in- cubat ion condit ions the rat adrenal enzyme dis- played more than twenty times lower apparent af-

10 -4

204 Naunyn-Schmiedeberg's Arch. Pharmacol. 294 (1976)

finity for ACTH1-a4. Addition of Gpp(NH)p during preincubation shifted the apparent Km value for the rat adrenal adenylate cyclase which was now similar to that seen in the absence of Gpp(NH)p with bovine adrenal cortex adenylate cyclase. The fragment ACTHl-17 had considerable intrinsic activity (0.8, if ACTH1-24 has 1.0) for the bovine adrenal cortex and somewhat less (0.6) for the rat adrenal enzyme. With Gpp(NH)p the fragment gained intrinsic activity for both enzyme preparations and stimulated adenyl- ate cyclase nearly to the same extent as did ACTH1-24 or ACTH1-23. The concentration-response curve for ACTH1 17 was bell shaped.

Similar curves have been reported for the response of intestinal adenylate cyclase to 7-oxa-13-prostynoic acid and were interpreted as interactions of the same effector molecule with two independent sites, mediat- ing opposite effects (Kantor et al., 1974). A survey of the literature reveals that apparent inhibition of adenylate cyclase by high concentrations of agonists is often observed but rarely discussed (see, however, Schramm and Rodbell, 1975 and for a general dis- cussion: Ariens, 1964). Unspecific effects of poly- cations (Wolff and Cook, 1975) and anions (Johnson et al., 1975) on adenylate cyclase have also been re- ported. Since an ACTH antagonist with high affinity was not available to us we cannot exclude the pos- sibility that some inhibitory contaminant is present in the peptide preparations or that the inhibition is mediated by a mechanism not involving receptors.

It is noteworthy to mention here, that ACTH1-16 is a full agonist in isolated rat adrenal cells for cyclic AMP production although with less apparent affi- nity than ACTH1-24. Very small increases of adenyl- ate cyclase activity were observed with ACTH6-17 and the rat adrenal enzyme when Gpp(NH)p was omitted; ACTHI-9 or ACTHI-~0 were completely inactive under these conditions. ACTH6-17 and the fragments ACTHI-9 and ACTHI-lO gained intrinsic activity in the presence of Gpp(NH)p. The three fragments were stimulatory without Gpp(NH)p for the bovine adrenal cortex adenylate cyclase system. The question arises in this context what factors determine "in- trinsic activity" and "apparent affinity" of hormone fragments and analogues and next, why the rat adrenal enzyme behaved differently from the bovine adrenal cortex enzyme. One can assume that the hormone receptor upon occupation by an agonist undergoes a conformational change, the magnitude of which is determined by the ligand. Somehow this signal is propagated by a coupling process involving guanylnucleotide sites to the catalytic subunits of the adenylate cyclase complex.

The final response of adenylate cyclase will there- fore not only depend on the fractional occupancy

of the hormone receptor but also on the coupling process. The theoretical aspects of this problem are similar to those regarding the relation of "stimulus" and "effect" in classical pharmacology as discussed by Ariens (1964) and more recently by Rodbard (1973). Jard et al. (1975) have speculated that the hormone receptor may lead to the formation of an intermediary product which is proportional to the degree of receptor occupancy and furthermore that the proportionality constant relating intermediate formation to receptor occupancy depends on the nature of the agonist. If adenylate cyclase activation is a saturable function of the intermediate the final degree of adenylate cyclase activation will depend on the affinity of the intermediary product to ade- nylate cyclase, the fractional occupancy of the receptor and the proportionality constant. Although there is little evidence that this model is correct it illustrates the complexity of the hormonal response even in an in vitro system. Within the frame work of this model one can assume that stimulatory guanylnucleotides increase the affinity of adenylate cyclase to the proposed intermediate. Gpp(NH)p, being resistant to hydrolysis by nucleotide phosphohydrolases, can effectively substitute for GTP and can even convert the activation by hormones from a reversible to an irreversible process (Schramm and Rodbell, 1975).

It would be a logical consequence of this and simi- lar models that "partial" agonists (characterized by a lower rate of intermediate formation at the same degree of fractional occupancy compared with the full agonist) can be converted to "full agonists" if the coupling process is changed to a favourable direc- tion e.g. by addition of Gpp(NH)p.

It is difficult to answer the question why the bovine adrenal cortex enzyme behaved differently from the rat adrenal enzyme. It has been shown for the rat adrenal enzyme that loosely bound stimulatory gu- anylnucleotides contaminate the enzyme preparation (Londos and Rodbell, 1975). Neither dialysis nor extensive washings were able to reduce the response of bovine adrenal cortex membranes towards ACTHl-24. It can, however, not be excluded that tightly bound guanylnucleotides are present to a different extent in both preparations. Alternatively it should be considered that both membrane prepara- tions differ in their lipid composition. Orly and Schramm (1975) recently reported that unsaturated fatty acids can enhance the activation of turkey eryth- rocyte adenylate cyclase by isoproterenol. Addition of free fatty acids to intact erythrocytes also affected the response of adenylate cyclase in membranes pre- pared from these pretreated cells.

As discussed above, addition of Gpp(NH)p con- verted the fragment ACTHl-17 to a full agonist for

H. Glossmann and C. J. Struck: In vitro Activity of ACTH Fragments 205

both adenylate cyclase systems and revealed the in- trinsic activity of ACTHt-9 and ACTH1 10 to stimu- late rat adrenal adenylate cyclase, mimicking the situation found in vivo. 2 This appears to be in con- trast to the results obtained with the nitrophenyl- sulfenyl analogue of ACTH1-24. Trp(Nps) 9 ACTH1-24 increases cyclic AMP production in iso- lated rat adrenal cells only to a very limited extent (Seelig and Sayers, 1973; Moyle et al., 1973). The rat adrenal adenylate cyclase system responded to the analogue in a similar way when Gpp(NH)p was omitted from the assay medium. Addition of Gpp(NH)p increased the intrinsic activity of the anal- ogue to about 0.25 and Trp(Nps) 9 ACTHt-24 was converted to a partial agonist. Since the analogue is a partial agonist (intrinsic activity 0.75) for the functional response, there appears to be a better corre- lation between the ability of Trp(Nps) 9 ACTH1-2~ to enhance the action of Gpp(NH)p in vitro and the functional response in vivo. It could be argued that the use of the artificial ligand Gpp(NH)p greatly amplifies the signal from receptors occupied by Trp(Nps) 9 ACTH1-24 under in vitro conditions. In vivo this signal may be never or only very weakly propagated to the catalytic subunits of adenylate cyclase. Exactly the opposite must then be postulated to explain the findings with some ACTH fragments and rat adrenal adenylate cyclase. Our findings can be summarized as follows: Differences of the in vitro response between the rat and bovine adrenal adenylate cyclase were minimized when Gpp(NH)p was added to the former preparation. By manipulation of the in vitro conditions to assay adenylate cyclase "partial" agonists could be converted to "full" agonists. A hormone analogue demonstrated considerable in- trinsic activity to stimulate rat adrenal adenylate cyclase-an observation which appears to be in con- trast to experiments with intact rat adrenal cells. We assume that the receptors for ACTH are very similar in both target tissues but that other factors (not necessarily guanylnucleotides) determine the hormo- nal response of adenylate cyclase in vitro and possibly in vivo as well.

Acknowledgement. We would like to thank those who donated peptides and K. Klappetek who provided excellent technical as- sistance. Deutsche Forschungsgemeinschaft supported us with a grant.

REFERENCES

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2 ACTH~ lo increases cyclic AMP in isolated adrenal cells (Seelig and Sayers, 1973).

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Received January 5 / Accepted May 4, 1976