The effects of Ca2+ and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

331

Biochimica et Biophysica Acta, 633 (1980) 331--346 @)Elsevier/North-Holland Biomedical Press

BBA 29465

THE E F F E C T S O F Ca 2+ AND GUANYLNUCLEOTIDES ON ISOPRENALINE-STIMULATED CYCLIC AMP FORMATION IN RAT RETICULOCYTE GHOSTS

H A R T M U T PORZIG, MARTIN SCHNEIDER and HELENA E. MAKULSKA *

Pharmakologisches Institut der Universit~'t Bern, Friedbiihlstrasse 49, CH-3010 Bern (Switzerland) (Received March 7th, 1980) (Revised manuscript received August 19th, 1980)

Key words: Ca2+; Guanylnucleotide; cyclic AMP accumulation; Isoprenaline stimulation; (Rat reticulocy te ghost)

Summary We have studied ~-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5 , 8 nmol/min per ml ghosts and remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (Ka) for (-+)-isoprenaline from 0.1 to 0.6 pM. The apparent dissociation constant for propranolol (0.01 ~M) remained unchanged. The K a values for isoprenaline in native reticulocytes and in resealed ghosts were identical. The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal/]-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca 2+ concentrations ranging between 0.1 and 1 #M. GTP stimulated isoprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3--5 but did not change the Ka value for isoprenaline. K a values for the guanylnucleotides in different experiments varied between 0.3 and 2 p_M. Ca 2÷ concentrations up to 4.6 pM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their K a values. * Present address: Medical A c a d e m y Warsaw, Institute of Physiological Sciences, Warsaw, Poland. Abbreviations: Hepes, N-2-Hydroxyethylpiperazine-N~-2-ethane sulphonic acid; DAPP, pI,pS.di(adenosine-5 -)pentaphosphate; [ Ca]i, [Ca] o, Ca2+ concentrations in the intracellular or i n c u b a t i o n m e d i u m respectively.

332 Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native ~-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are n o t observed in isolated membranes.

Introduction Several recent studies have shown that Ca 2+ or GTP modify the activity of the H-receptor-coupled membrane enzyme adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) in a variety of cells. However, nearly all of the pertinent results have been obtained from broken membrane preparations [1--4]. Rather limited information is available on the function of this system in the intact cell, i.e. under conditions where the natural spatial organization of its components is maintained and the enzyme moiety is kept in contact with the cytosol. In most cells the composition of the intracellular medium cannot bc controlled satisfactorily. In particular, components of the cytosol which are known or suspected to regulate the hormone-stimulated enzyme activity such as guanylnucleotides have a very low membrane permeability and will penetrate poorly if applied from the outside. Some obvious discrepancies between the behaviour of the intact cell and experimental findings in the corresponding membrane preparations suggest that the isolation of plasma membranes may introduce quantitative and qualitative changes in the reaction of the hormone-sensitive enzyme towards activating or inhibitory stimuli. (a) Half-maximal activation of adenylate cyclase by f~-adrenergic agonists in broken membranes often requires concentrations one order of magnitude above those needed in the intact cell [5--7]. In some cases the fi-receptor-mediated .stimulation of the enzyme is completely lost [8,9]. (b) Fluoride is a strong activator of adenylate cyclase in membranes but is usually inactive in native cells [10]. (c) In membrane preparations, guanylnucleotides usually activate basal and ~-agonist-stimulated adenylate cyclase and tend to decrease agonist affinity for f~-receptors [7] b u t their role in the intact cell is controversial [11--13]. (d) Desensitization of adenylate cyclase towards ~-adrenergic agonists associated with a reduction in/~-receptors numbers can be induced only in intact cells b u t n o t in membrane preparations [14]. We have studied isoprenaline-stimulated cyclic AMP formation in unsealed and in resealed ghost cells prepared from rat reticulocytes as model systems intermediate between intact cells and broken membranes. Preceding experiments have shown that the membrane permeability of resealed reticulocyte ghosts to Ca ~÷, K ÷ or Na ÷ and the adenyl- or guanylnucleotides was comparable to that in intact cells and that the composition of the intracellular medium in ghosts could be manipulated within wide limits [ 15--17]. The ~-adrenergic system of rat reticulocyte membranes has been studied previously by Palm and coworkers [5,18,19] and by Bilezikian et al. [6,20]. Their work provides a valuable frame of reference for our results using ghost

333 cells. The hormone-sensitive adenylate cyclase activities in reticulocyte membranes and in comparable preparations from many other tissues share the sensitivity towards guanylnucleotides, the stimulatory effect of NaF and the inhibitory action of Ca. Our results suggest that the interaction of intracellular Ca2÷ and guanylnucleotides with the ~-adrenergic system in ghosts and intact cells differs considerably from their mode of action in membrane preparations. Materials and Methods Rats (mostly female), weighing 150--300 g (SIV 50 breed/Tierzucht Ziirich and Wistar rats, Pathophysiologisches Institut, University of Bern) were used. Hemolytic anaemia was induced by intramuscular injection of 30 mg/kg acetylphenylhydrazide for three consecutive days [5]. On the 7th day after the first injection the rats were decapitated and the blood, containing 40--60% reticulocytes, was collected into 4% citrate solution. The blood was immediately illtered through nylon wool (Leuko Pak of Fenwall Laboratories, Deerfield IL, U.S.A.) to remove leukocytes and platelets. The red cells were then washed three times in isotonic NaC1 solution.

Preparation of reticulocy te ghosts Washed reticulocyte cells were subject to density gradient centrifugation in colloidal silica sol (Percoll® from Pharmacia AB, Uppsala, Sweden). The medium contained 50 mM Hepes and 120 mM KC1 (pH 7.0) to maintain a tonicity of 290 mOsm and was adjusted to an initial density of between 1.087 and 1.096. The final gradient developed during centrifugation of the cell suspension in Percoll for 25 min with 17 000 × g at 4°C in a fixed angle rotor (Sorvall RC2B centrifuge). This procedure yielded clearly separated cell bands. The top layer contained most of the reticulocytes, but virtually no old cells (characterized by intracellular Heinz bodies). This layer was carefully removed by suction and washed free of Percoll in isotonic saline. Cell ghosts were then prepared by reversal of osmotic hemolysis at 0°C, pH 7.0 essentially according to the method of Bodemann and Passow [21]. It was found that only reticulocytes and young erythrocytes could be transformed into resealed ghosts with this method. Old cells from the bottom layer of the gradient did not reseal upon reversal of osmotic hemolysis.

Preparation of ghost populations with different degrees of membrane resealing Like ghosts from mature human red cells [22] reticulocyte ghosts, after osmotic hemolysis, recovered a low membrane permeability for cations and nucleotides in a time- and temperature-dependent process. Therefore, cell populations differing in their permeability characteristics could be obtained by varying the duration of the resealing period. (a) Ghosts maintained at 0°C after re-establishing isotonicity do not reseal towards small molecules (molecular weight <5000) but keep an erythrocyte-like shape and normal membrane orientation. We call this preparation 'unsealed ghosts'. (b) Incubation of ghosts in isotonic medium for 5--7 min at 37°C after reversal of hemolysis at 0°C, results in a mixed population of 30--50% sealed and 50--70% unsealed ghosts because the individual cells of a ghost population do not simultaneously

334 recover a low permeability to small molecules [21]. We call this preparation 'partially sealed ghosts'. (c) After an incubation period of 20 min at 37°C 90% of the ghosts are sealed to ATP and guanylnucleotides. 60 min were required to seal a majority of the ghosts to Na ÷ and K ÷. All ghost preparations allowed to recover for 20 min or longer are called 'resealed ghosts. The composition of the incubation medium during the resealing period determines the composition of the intracellular medium in sealed ghosts. In most ' experiments, resealed ghosts were prepared so that they contained ATP together with the ATP regenerating system, phosphocreatine (5--10 mM) and creatine kinase (14 U/ml) (soln. I or II in Table I). The same regenerating system was included in the incubation medium for experiments designed to assess the accessibility of adenylate cyclase for extracellular ATP. Moreover, in most experiments the cells also contained 20 #M DAPP, an inhibitor of adenylate kinase. This enzyme is present in red cell membranes and tends to lower the actual ATP concentration by establishing an equilibrium between ATP, ADP and AMP [23]. The cellular ATP content was checked under a variety of conditions using the firefly lantern assay as modified by Stanley and Williams [24]. The ATP concentrations in resealed ghosts in the presence of an ATP-regenerating system were approximately equal to extracellular concentrations during the resealing period. We assumed a similar distribution for GTP and Gpp(NH)p. A 10-fold or 100-fold dilution of the original cytosol was achieved by hemolyzing 1 vol. of cells in 10 or 100 vol. of hypotonic hemolyzing solution (tonicity 30--60 mOsm). Isotonicity was restored in the cell lysate by adding a 3 M KC1-Hepes solution buffered to pH 7.0 to make K ÷ the main intracellular cation. At the end of the variable resealing periods the ghosts were washed three times in KC1-Tris solution (120 mm KC1/50 mM Tris-HC1, pH 7.4) and resuspended in KC1-Hepes solution (120 mM KC1/50 mM Hepes, pH 7.0) to give a stock suspension with a cytocrit of 10--15%. The actual cytocrit value in each experiment was determined with conventional microhematocrit capillaries. In some experiments we prepared 'fragmented reticulocyte membranes' essentially as described b y Porzig and Stoffel [25]. The m e t h o d includes hypotonic hemolysis of reticulocytes followed by at least three consecutive freezethawing cycles. This treatment disrupts the cell membrane irreversibly. A stock suspension of membranes was prepared in KC1-Hepes solutions as above. Its protein c o n t e n t (usually 2--3 mg/ml) was determined by the biuret method.

Determination of cyclic AMP formation All assays were run in duplicate. For each sample the reaction was initiated by adding 200 ~l of the ghost cell or membrane stock suspension to 200 #1 of the experimental medium which contained KC1 as main osmotic constituent. Thus, cellular volume changes caused by an a s y m m e t r y in cation fluxes could be avoided. As ATP-free incubation medium we used solution V in Table I. ATPcontaining media corresponded mostly to solution IV containing 2 mM ATP and 3 mM MgC12. In experiments designed to test the effect of guanylnucleotides we used mostly solution VI containing 50 or 200 pM ATP. L o w ATP concentrations were used to reduce the interference from GTP as a contaminant of commercial ATP preparations. Further detail is given with individual experiments. After a predetermined time period at 37°C {usually 4 min) the reaction

335 TABLE

I

Composition o f s o l u t i o n s i s e x p r e s s e d i n r a M . A b b r e v i a t i o n s a r e as f o l l o w s : I B M X , i s o b u t y l m e t h y l x a n thine; CrP, ereatinephosphate; CPK, ereatinephosphokinase; BSA, bovine serum albumin; Special addt., special additives; h.m., hemolysing medium; i.m., incubation medium. Solution

KCI Hepes EGTA ATP Mg IBMX DAPP Special addt.

pH Use

No.

I

II

ni

iv

v

vI

-10 2 0.05--2.0 1.4--3.0 -0.02 10 CrP CPK (0.I mg/ml) Ouabain (10 -4 g/ml) 7.0 h.m.

20 10 2 0.05--2.0 1.4--3.0 -0.02 10 CrP CPK (0.I mg/rnl) Ouabain (10 -4 g/ml) 7.0 h.m.

20 10 2 -1.5 ----

125 35 1 0.05--2.0 1.4--3.0 1 ---

125 35 1 -1.5 1 ---

7.0 h.m.

7.0 i.m.

7.0 i.m.

125 35 1 0.05--2.0 1.4--3.0 1 0.02 5--10 CrP CPK (0.I mg/ml) BSA (1 m g / m l ) 7.0 i.rn.

was stopped b y heating the closed polypropylene reaction vessels in an aluminium block maintained at 100°C, for 4 min. The samples were then sedimented in an Eppendorf Laboratory Centrifuge (type 3200) at 14 000 × g for 1 min. Total cyclic AMP formation, i.e., the sum of extracellular and intracellular cyclic AMP was determined after suitable dilution in the protein-free supernatant of the samples according to the m e t h o d of Tovey et al. [26]. Calibration curves for cyclic AMP {0.5--16 pmol/sample) set up either in buffer solution or in heat-deproteinized supernatant did n o t differ significantly. Samples of protein-bound 3H-labelled cyclic AMP were dissolved in 10 ml scintillation fluid containing a 1 : 1 (v/v) mixture of Triton X-100 with xylol and 4 g/1 Omnifluor ® (New England Nuclear). 3H was counted in an Intertechnique SL 4000 liquid scintillation counter. Most experimental incubations were performed in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. With a maximally effective concentration (1 mM) cyclic AMP accumulation in the system was linear for at least 15 min. The rate of cyclic AMP formation was always related to the known volume of packed ghost cells in each sample. It is important to note that the yield in ghost volume with respect to the original cell volume was similar for all types of ghost.

Evaluation o f data Values for half-maximal stimulation of adenylate cyclase by isoprenaline or guanylnucleotide (Ka values) were evaluated graphically from concentrationresponse curves. The apparent dissociation constants (Kd) for the ~-adrenergic antagonist propranolol were either calculated from the shift of the isoprenaline concentration-response curves or were evaluated graphically from a Schild plot [27]. Student's t-test was used for statistical analysis of the data.

336 Estimation o f Ca 2÷ and Mg 2÷ concentrations Ca 2÷ concentrations in CaEGTA-buffer solutions were calculated according to Porzig and Stoffel [25] using equations given by Portzehl et al. [28]. We assumed a pK' value of 6.62 for the Ca-EGTA complex at pH 7.0. A more detailed description of the procedure and an account of the criteria for choosing particular pK values is given by Porzig and Stoffel [25,29]. For the calculation of free Mg 2÷ and Ca 2÷ concentrations in the presence of two complexing agents (ATP and EGTA) a system of eight simultaneous equations was arranged such that it could be solved by an iterative computer program on a HewlettPackard 9010 desk-top computer. The calculations showed that the presence of Mg 2÷ (1.5 mM) had very little effect on the EGTA-buffered free Ca concentrations. The same was true for the effect of Ca and EGTA on free Mg 2÷ and free ATP (HATP3-+ ATP 4-) concentrations. Therefore free Mg 2÷ and free ATP could be estimated with reasonable precision even if the simultaneous presence of the Ca/EGTA system were disregarded. Mate rials All chemicals used in the experiments were of the highest purity commercially available. Reagents and sources: EGTA, phosphocreatine, creatine kinase, cyclic AMP-dependent protein kinase, firefly lanterns, Hepes, isobutylmethylxanthine (Sigma Corp. St. Louis, MO, U.S.A.); ATP, cyclic AMP, DAPP, GTP, Gpp(NH)p, adenosine (Boehringer Corp. Mannheim, F.R.G.); 1-acetyl-2phenylhydrazide (Schuchart, Mfinchen, F.R.G.). In part of the experiments we used a cyclic AMP assay kit based on the m e t h o d of Tovey et al. [26] of the Radiochemical Center, Amersham, U.K. From the same source we obtained 3H-labelled cyclic AMP. (-+)-Propranolol was a gift of ICI, Macclesfield, U.K. The antibiotic divalent cation ionophore A 23187 was a gift of Eli Lilly (Dr. L. Hamill), Indianapolis, U.S.A. (±)-Isoprenaline was Pharmacopoea Helvetica grade. Results

Effect o f membrane recovery on isoprenaline-stimulated cyclic A M P formation In the first set of experiments we studied the rate of cyclic AMP formation supported by intra- or extracellular ATP during transition from unsealed to resealed ghosts (Table II). All experiments were performed in the presence of isobutylmethylxanthine to suppress phosphodiesterase activity. All incubation media contained 10 #M isoprenaline. In unsealed ghosts, more than 95% of isoprenaline-activated adenylate cyclase activity was accessible to extracellular ATP if free Ca 2÷ in the incubation medium was kept below 10 -8 M by means of EGTA buffering. In partially sealed ghosts, 30--60% of the total receptor-coupled enzyme activity was located in cells impermeable to ATP. In resealed ghosts the formation of cyclic AMP depended almost exclusively on intracellular ATP. When ATP was present simultaneously on both sides of the membrane, total isoprenaline-stimulated cyclic AMP accumulation usually equalled the sum of cyclic AMP formed in the presence of either intraceUular or extracellular ATP. Therefore, no substantial leakage of ATP appeared to occur from sealed ghosts into the medium or

337 TABLE

II

RATE OF ISOPRENALINE RETICULOCYTE GHOSTS ABSENCE O F Ca 2+

(Ipn)- O R F L U O R I D E - S T I M U L A T E D cAMP RESEALED FOR DIFFERENT TIME PERIODS

FORMATION IN R A T IN T H E P R E S E N C E OR

T h e c e l l s w e r e l o a d e d w i t h 2 m M E G T A ( [ C a ] i ~ 1 0 - 8 M ) o r a C a / E G T A b u f f e r m a i n t a i n i n g f r e e [ C a ] i at 4 . 6 • 1 0 - 6 M. I n c u b a t i o n ( 4 rain) o f A T P - l o a d e d g h o s t s i n t h e p r e s e n c e o r a b s e n c e o f 2 m M A T P ( s o l n . I V o r V , r e s p e c t i v e l y ) . A T P - f r e e g h o s t s w e r e i n c u b a t e d in t h e p r e s e n c e o f 2 r a M e x t r a c e l l u l a x A T P ( s o l n . I V ) . All A T P m e d i a c o n t a i n e d 3 m M Mg. T h e i n c u b a t i o n m e d i u m c o n t a i n e d 1 0 p M i s o p r e n a l i n e o r 1 0 m M N a F . T h e values r e p r e s e n t t h e m e a n ± S . E . o f 2 - - 1 0 i n d e p e n d e n t e x p e r i m e n t s ( n u m b e r o f e x p e r i m e n t s in b r a c k e t s ) . n . s . , n o t s i g n i f i c a n t ; i., i n s i d e ; o . , o u t s i d e ; e . , e i t h e r s i d e . P v a l u e s a n d t h o s e m a r k e d n.s. are as c o m p a r e d 1 5 t h e r e s p e c t i v e v a l u e s for 0 rain r e s e a t i n g in t h e a b s e n c e o f Ca. Resealing time (min)

0

Cahe m (M)

10 -8

Location of substrate (ATP)

Rate of cAMP formation (pM/min per ml ghosts) 1 0 -5 M I p n a d d e d

10 -2 M N a F added

i. o. e.

70 (2) 2 0 2 5 + 4 6 8 (4) 2406 ± 117 (10)

897 ± 112 (3)

0

4.6 • 10 -6

i. o. e.

2231 1 0 4 0 +- 2 8 0 3 8 5 4 -+ 3 8 8

(2) (3) ( 5 ) **

875

5

10 -8

i. o. e.

1 7 5 3 -+ 2 2 0 2 4 7 1 -+ 5 7 4 3 7 5 8 +- 5 0 4

(8) (6) (8) *

630 ± 142 (4)n.s.

5

4.6 • 10 -6

i. o. e.

4 4 7 6 +- 9 1 7 526 ± 97 4 3 3 0 -+ 5 9 1

(7) (4) (7)

7 2 0 +- 9 6 ( 4 )

10 -8

i. o. e.

4206 ± 523 413 3 4 2 7 -+ 4 8 9

(4) (2) (6)

5 2 0 ± 1 1 6 ( 3 ) n.s.

60

(2)

* P ~ 0.02. ** P < 0 . 0 0 1 .

vice versa. The actual amount of ATP trapped in ghosts after resealing periods of 0, 5 or 60 min in the presence or absence of Ca 2÷ was measured by the firefly lantern assay. From the ratio between cellular ATP and total ATP concentration in the hemolyzing medium, the fraction of ghosts that had actually sealed for ATP was calculated. The percentage of ATP sealed cells in the unsealed or partially sealed ghost preparation was in good agreement with the fraction of total cyclic AMP formation supported by intracellular ATP (estimated from Table II). Apparently, most of the cells that sealed for ATP could be stimulated by isoprenaline and hence must have kept their original outside-out orientation. After a 5 min resealing period in a Ca-free medium the maximal rate of cyclic AMP formation in the presence of isoprenaline increased significantly from 2.4 to 3.7 nmol/ml ghosts per min. A similar increase was obtained without a resealing period if ghosts were prepared in the presence of /~M concentrations of Ca 2÷. However, the effects of a resealing period and of Ca 2÷ on the rate of cyclic AMP formation were not additive. Moreover, Ca 2÷ also accelerated the resealing towards ATP in the cold (Table II). Therefore, the increase in cyclic AMP formation probably reflected the recovery of membrane integrity rather than a direct stimulating effect of Ca.

338 %

//i-2

~ 50 i ~ ,~

0 i"

I0-9

I0"e

dO-7

i0-6

reseoled ghosts fragmented membrones I0 -5

i0-4

Isoprenoline (M) A 4

% ~-

E~

o reseoled ghosts intact ceils

o L ~ a-2

~

/

tO0

.:. o

o

~

!ntact ceils

80

~ g

<~ ~

's

60 reseoled ghosts [ 40

0

•

0

10-8 10-7 I0~6 10-5 Isop,enollne (U)

~

0

4~ 10-4

-/0-3 [Co++]o(M )

• [0-2

Fig. 1. E s t i m a t i o n o f K a v a l u e s f o r i s o p r e n a l i n e t o s t i m u l a t e cyclic AMP ( c A M P ) f o r m a t i o n in f r a g m e n t e d m e m b r a n e s , r e s e a l e d g h o s t s a n d n a t i v e r e t i c u l o c y t e s . A: C o m p a r a t i v e c o n c e n t r a t i o n - r e s p o n s e c u r v e s for i s o p r e n a l i n e in f r a g m e n t e d m e m b r a n e s (o o, n = 4), a n d in r e s e a l e d g h o s t s l o a d e d w i t h 2 m M A T P / 3 m M MgC12 (e e, n = 7). T h e i n c u b a t i o n m e d i u m (soin. I V ) c o n t a i n e d t h e s a m e A T P a n d Mg conc e n t r a t i o n as the i n t r a c e l l u l a r m e d i u m a n d w a s i d e n t i c a l f o r m e m b r a n e s a n d ghosts. Bars give -+S.E. Isop r e n a l i n e K A v a l u e in m e m b r a n e s , 6.3 • 10 -7 M; in r e s e a l e d ghosts, 1 • 10 -7 M. B: C o m p a r a t i v e c o n c e n t r a t i o n - r e s p o n s e c u r v e s f o r l s o p r e n a l i n e in n a t i v e r e t t c u l o c y t e s a n d g h o s t s f r o m t h e s a m e b l o o d s a m p l e in one e x p e r i m e n t . G h o s t s p r e p a r a t i o n similar to t h a t in A. N a t i v e cells w e r e i n c u b a t e d in a s o l u t i o n c o m p o s e d of ( r a M ) 1 3 0 KC1/20 H e p e s / 0 . 5 E G T A / 2 MgC]2/1 i s o b u t y l m e t h y l x a n t h i n e / 5 g l u c o s e / 1 0 inosine. Isop r e n a l i n e K a v a l u e in i n t a c t cells; 4 . 4 • 1 0 -8 M, in ghosts: 5.2 • 10 -8 M. Fig. 2. I n h i b i t o r y e f f e c t o f e x t r a c e l l u l a r Ca 2+ on i s o p r e n a l l n e - d e p e n d e n t cyclic AMP ( c A M P ) f o r m a t i o n in r e s e a l e d g h o s t s a n d in i n t a c t r e t i c u l o c y t e s . G h o s t s w e r e p r e p a r e d to c o n t a i n 2 m M A T P a n d 0.7 m M MgC12. I n c u b a t i o n (4 r a i n ) o f g h o s t s in s o l u t i o n V c o n t a i n i n g u n b u f f e r e d Ca 2+ c o n c e n t r a t i o n s r a n g i n g f r o m 0 . 5 to 10 m M a n d 1 0 / ~ M i s o p r e n a l i n e . T h e e x p e r i m e n t a l p o i n t s r e p r e s e n t n o r m a l i z e d m e a n v a l u e s -+S.E. of 5---6 e x p e r i m e n t s . I n t a c t cells w e r e e x p o s e d f o r 4 rain to t h e s a m e Ca 2+ a n d isoprenalLne c o n c e t r a t i o n s , i n c u b a t i o n m e d i u m o t h e r w i s e similar as in "Fig. 3B. E x p e r i m e n t a l p o i n t s give t h e m e a n of duplicate d e t e r m i n a t i o n s in o n e e x p e r i m e n t .

It is impossible to decide from the experiments presented in Table II which of the partial functions of the t-receptor adenylate cyclase system are changed during the resealing process. In subsequent experiments we measured Ka values for isoprenaline and K d values for propranolol in different states of membrane recovery. In Fig. 1A we have plotted concentration-response curves for isoprenaline obtained from fragmented reticulocyte membranes and from resealed reticulocyte ghosts. The mean Ka value for (±)-isoprenaline in ghosts (1.01 ± 0.23 • 10 -7 M) was significantly lower than in isolated membranes (6.3 ± 1.3 • 10 -7 M). In partially sealed ghosts the agonist K a values tended to be lower in the sealed than in the unsealed fraction. However, this difference was not statistically significant. Basal cyclic AMP formation in the absence of isoprenaline was 7.8 ± 1.2% of the maximal fl-adrenergic response in ghosts but was 18.5 ± 4.9% in isolated membranes. In Fig. 1B the concentration-response curve for isoprenaline-activated cyclic AMP synthesis in resealed ghosts is compared to the corresponding curve in intact reticulocytes from the same sample of blood.

339 The Ka values for isoprenaline were almost identical. It is also obvious from Fig. 1B that cyclic AMP formation in resealed ghosts and in intact cells could be stimulated to the same extent. Hill plots for isoprenaline concentrationresponse curves in resealed ghosts loaded with 2 mM ATP and 3 mM MgC12 yielded a mean slope of 0.96 -+ 0.06 (n = 7), b u t the Hill coefficient in fragmented membranes was significantly lower than unity (0.75 -+ 0.05, n = 4). No significant shift in antagonist binding constants was associated with membrane reconstitution. Under all conditions we found Kd values for (+)-propranol close to 10 -a M (1.06 + 0.17 • 10-a).

NaF-dependent adenylate cyclase stimulation in reticulocyte ghosts It is well d o c u m e n t e d that the adenylate cyclase of intact cells usually cann o t be stimulated b y NaF, whereas in membrane preparations fluoride is generally a more p o t e n t stimulant than/3-adrenergic agonists [10]. The effect of NaF on cyclic AMP formation in different ghost preparations was therefore studied as an additional test for the degree of membrane recovery which can be achieved in resealed ghosts Table II. Even in unsealed ghosts, cyclic AMP formation in the presence of 10 mM NaF was only 33% of the value achieved with isoprenaline. In resealed ghosts NaF-mediated cyclic AMP accumulation decreased to 15% of maximal/3-receptor-mediated stimulation. Such a decrease was observed in each of three independent experiments. However, the absolute cyclic AMP response to NaF stimulation was variable and the mean decrease from 807 to 520 pmol/min per ml ghosts (Table II) was not statistically signicant.

The inhibitory effect of Ca on cyclic AMP formation It is possible that the sensitivity of the adenylate cyclase for Ca 2+ [30--33] is also modified by the functional state of the membrane. We have compared the effect of Ca on cyclic AMP formation in intact cells, in fragmented membranes and in ghosts which were prepared in the absence of Ca. In the experiments as shown in Fig. 2, intact cells or resealed ghosts were exposed to extracellular Ca concentrations between 0.5 and 10 mM. Isoprenaline-stimulated cyclic AMP formation was inhibited by 20% with 1 mM Ca in ghosts and with 3.5 mM in native cells. In control experiments the same Ca concentrations had no effect or. the K a value of isoprenaline. Thus, the inhibitory effect of Ca 2÷ was n o t caused by a progressive shift of the concentration-response curves for/3-adrenergic stimulation, b u t was probably due to an increase in [Ca] i. R a t reticulocytes possess an outwardly directed active Ca 2÷ transport mechanism which is similar to that of human red cells, and is maximally activated with 2--3 pM [Ca]i (Makulska,' H.E. and Porzig, H., unpublished results}. Surprisingly, this Ca p u m p could n o t maintain a stationary low value of [Ca]i in the presence of moderately increased levels of extracellular Ca 2÷. This finding could be explained if [Ca]i values below the K m value of the p u m p (approx. 10 -6 M) would be inhibitory for the adenylate cyclase. The Ca sensitivity of adenylate cyclase in unsealed, partially sealed or resealed ghosts, was tested by pre-equilibrating the cells with EGTA-buffered Ca 2÷ concentrations ranging from 10 -8 to 4.6 • 10 -6 M in the presence of the divalent cation ionophore A 2 3 1 8 7 (2 ~M) [34] {Fig. 3). ATP (2 mM) and Mg ~÷

340 '~

150

bOO

~ 80 EO

o

/,

o a- ~

/ / Gpp(NH)p +L

60

D

~ ~0

ghosts

~(~.l

o

0

•

..

0

iO-r

['--~ ~ i ; IO-S

[co"}o (M)

reseohng time

I eoled • unsealed 0 frogmenfed membrones 10-5

E

_ E & ~,

//

50

~ o

cL

~ o

~ _ 0

f/

GTP (b)

I0 ; i0 -6 IO -5 I0 -4 I0 3 GTP or Gpp{NH)p (M)

Fig. 3. C a - d e p e n d e n t i n h i b i t i o n of i s o p r e n a l i n e - s t i r n u l a t e d cyclic AMP ( c A M P ) f o r m a t i o n in f r a g m e n t e d m e m b r a n e s a n d r e t i c u l o c y t e ghosts. F r a g m e n t e d m e m b r a n e s w e r e i n c u b a t e d in s o l u t i o n I V c o n t a i n i n g 2 m M E n T A o b u f f e r e d tree Ca 2+ c o n c e n t r a t i o n s r a n g i n g f r o m 10 - 8 t o 4.6 • 10 - 6 M, 2 m M A T P , 3 raM MgC12 a n d 10 # M i s o p r e n a l i n e . E x p e r i m e n t a l p o i n t s f o r f r a g m e n t e d m e m b r a n e s f r o m one o f t h r e e similar b u t less c o m p l e t e e x p e r i m e n t s . U n s e a l e d , p a r t i a l l y sealed a n d r e s e a l e d g h o s t s w e r e p r e p a r e d in t h e prese n c e of 2 m M A T P a n d 3 m M MgC12. T h e g h o s t s w e r e p r e i n c u b a t e d in s o l u t i o n V c o n t a i n i n g 2/~M i o n o p h o r e A 2 3 1 8 7 b u t n o d i v a l e n t c a t i o n s . A f t e r 2 rain A T P , MgC12 a n d Ca 2+ or C a - E G T A b u f f e r s w e r e a d d e d (final c o n c e n t r a t i o n s : 2 m M A T P , 3 m M MgC12, 10-8---4.6 • 10 -6 M Ca 2+ ( b u f f e r e d ) 1 0 - 4 - - 1 0 -3 Ca 2+ ( u n b u f f e r e d ) . A f t e r a f u r t h e r 3 rain i s o p r e n a l i n e was a d d e d (final c o n c e n t r a t i o n 10 #M). Cyclic AMP w a s m e a s u r e d 4 rain a f t e r a d d i t i o n o f i s o p r e n a l i n e . E x p e r i m e n t a l p o i n t s for g h o s t s give m e a n v a l u e s of f o u r d e t e r m i n a t i o n s in t w o i n d e p e n d e n t e x p e r i m e n t s w i t h i d e n t i c a l results. I n s e t : t i m e d e p e n d e n c e of t h e i n h i b i t o r y a c t i o n of [Ca] o o n i s o p r e n a l i n e - d e p e n d e n t cyclic A M P f o r m a t i o n of resealed r e t i c u l o c y t e g h o s t s in t h e p r e s e n c e o f A 2 3 1 8 7 . T h e o r e t i c a l [Ca] i a f t e r e q u i l i b r a t i o n of i n t r a - a n d e x t r a c e U u l a r c o n c e n txations w a s 0.8 raM. M e a n s o f d u p l i c a t e d e t e r m i n a t i o n s in one e x p e r i m e n t . Fig. 4. E f f e c t o f g u a n y i n u c l e o t i d e s a n d o f cell l y s a t e o n basal a n d i s o p r e n a l i n e - s t i m u l a t e d cyclic AMP ( c A M P ) f o r m a t i o n b y f r a g m e n t e d r e t i c u l o c y t e m e m b r a n e s ( 1 . 4 m g p r o t e i n / m l ) . T h e m e d i u m (soln. V I ) c o n t a i n e d 0.2 m M A T P a n d 1.6 m M Mg. 10 rain i n c u b a t i o n p e r i o d s in t h e p r e s e n c e or a b s e n c e of 1 0 0 #M i s o p r e n a l i n e w e r e s t a r t e d b y a d d i t i o n o f m e m b r a n e s to a p r e - w a r m e d m e d i u m . B r o k e n lines give the results o f parallel i n c u b a t i o n s o f t h e s a m e m e m b r a n e s u s p e n s i o n in t h e m e d i u m s u p p l e m e n t e d w i t h a 1 : 50 d i l u t i o n o f fresh r e t i c u l o c y t e l y s a t e (L). I s o p r e n a l i n e - s t i m u l a t e d cyclic AMP f o r m a t i o n is t h e diff e r e n c e b e t w e e n t o t a l a n d basal (b) cyclic A M P p r o d u c t i o n . M e a n o f d u p l i c a t e d e t e r m i n a t i o n s in one e x p e r i m e n t . T h r e e o t h e r e x p e r i m e n t s y i e l d e d similar results.

(3 mM) were maintained at similar concentrations on both sides of the membrane. It was essential to equilibrate the cells with the ionophore prior to the addition of divalent cations to observe the maximal inhibitory effect of a given Ca 2÷ concentration. The inset in Fig. 3 shows that the cells maintained a substantial rate of cyclic AMP synthesis for almost 10 min if Ca 2÷ and ionophore were added simultaneously rather than in succession. The slow kinetics of membrane partitioning and uptake of the Ca or Mg ionophore complex explains this behaviour [35]. Isoprenaline-stimulated cyclic AMP synthesis in fragmented reticulocyte membranes was half-maximally inhibited with 1 . 3 . 1 0 - 7 M Ca 2÷ (left-hand curve in Fig. 3). The Ca 2÷ concentrations required for 50% inhibition of cyclic AMP formation in ghosts appeared to increase from 2 . 2 - 1 0 -7 M (unsealed ghosts), to 4 - 1 0 - ? M (partially sealed ghosts), or to 6 . 2 . 1 0 -7 M (resealed ghosts). Probably, this finding does not reflect an increasing resistance of the adenylate cyclase towards Ca-mediated inhibition. Ferreira and Lew [36] have shown that at low values of [Ca]o the Ca pump of human erythrocytes will maintain an inwardly directed Ca concentration gradient even in the presence

341 of an ionophore-induced Ca leak. In ghosts, the pump-induced difference b e t w e e n [Ca]i and [Ca]o will b e c o m e progressively larger as the resealing proceeds. Thus, the shift to the left of the curves in Fig. 3 may merely reflect the improved efficiency in the outward pumping of Ca.

The effect o f GTP and Gpp(NH)p on isoprenaline-stimulated adenylate cyclase in reticulocyte ghosts The question of whether the pronounced regulatory effects of guanylnucleotides on the /~-receptor-coupled adenylate cyclase in membrane preparations give a true picture of their role in the intact cell is still unresolved [11,12,37, 38]. We have studied the effects of intracellular guanylnucleotides on isoprenaline-stimulated cyclic AMP accumulation in reticulocyte ghosts and in fragmented reticulocyte membranes. The membranes were prepared in solutions containing EGTA and less than 10 -s M free Ca 2+. The membrane preparation was then incubated in solution VI in the presence or absence of a maximally effective isoprenaline concentration (100 pM). GTP or Gpp(NH)p concentrations ranged between 0.3 and 500 #M. Basal adenylate cyclase activity was increased more than 20-fold b y Gpp(NH)p b u t was barely affected by GTP (2-fold increase). GTP and Gpp(NH)p p r o m o t e d fl-adrenergic cyclase stimulation in a similar concentration range (K a ~ 0.5 #M) b u t enhanced the maximal effect of isoprenaline by factors of 5.8 and 13, respectively. However, the K a value for isoprenaline as estimated from concentration-response curves did n o t change significantly in the presence of 100 #M GTP. Subsequently the action of GTP and Gpp(NH)p on basal and isoprenalinestimulated cyclic AMP formation in unsealed ghosts was studied (Figs. 4 and 5). The cells were prepared in the presence of EGTA and were incubated in media similar to those used for fragmented membranes. GTP and Gpp(NH)p increased basal cyclic AMP formation by factors of 2-+ 0.4 and 7.8-+ 2.1, respectively. With maximally effective concentrations of GTP and Gpp(NH)p basal cyclic AMP formation was 4.9 + 1.2% and 24.9 -+ 6.3% of total production in the presence of 10/xM isoprenaline (n = 4). In Fig. 5 the isoprenalinestimulated fraction of total cyclic AMP accumulation in a representative experiment is shown as a function of GTP or Gpp(NH)p concentration. The rate of cyclic AMP formation in the absence of guanylnucleotides and their absolute stimulatory effects were rather variable b u t the qualitative pattern was perfectly reproducible. With 0.2 mM ATP and 1.6 mM Mg in the incubation medium, 530 +- 164 pmol cyclic AMP/ml cells p e r min (n = 7) were formed in the presence of isoprenaline (10 pM) alone. The production rate was raised to 2052 -+ 463 pmol cyclic AMP./ml cells per min by GTP in = 4) and to 1421 -+ 161 pmol cyclic AMP/ml cells per min by Gpp(NH)p (n = 6). The relative stimulating effects of GTP in fragmented membranes and in unsealed ghosts were of comparable magnitude. However, Gpp(NH)p stimulated less efficiently than GTP in ghosts and compared to membranes its relative stimulating power was reduced by more than 80%. The apparent decrease in sensitivity of adenylate cyclase to Gpp(NH)p was n o t due to a shift in the concentrationresponse curve for isoprenaline. Such a shift has been observed in other cells [39]. The K~ value for the ~-agonist remained unchanged in the presence of 1 ~M GTP or Gpp(NH)p. Nevertheless, both nucleotides enhanced the rate

342 % 150,

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GTP

,,~ ,ntrocelkulo,

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(NH)p

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10-7 10-6 I0 -5 10-4 10-3 GTP or Gpp(NH)p (M)

0 I ~,. 0

I0 7 10-6 10-5 Gpp(NH)p Or GTP(M)

Gpp(NH)p ~ntracelluIQr

i0 -~,

F i g . 5. E f f e c t o f g u a n y l n u c l e o t i d e s and o f cell l y s a t e o n isoprenaline-stirnulated c y c l i c A M P ( c A M P ) prod u c t i o n in u n s e a l e d r e t l c u l o c y t e ghosts. G h o s t s w e r e i n c u b a t e d for 4 rain in s o l u t i o n VI, as d e s c r i b e d in the t e x t . F o r parallel i n c u b a t i o n s t e s t i n g the e f f e c t o f c y t o s o l the m e d i u m w a s s u p p l e m e n t e d w i t h a 1 : 5 0 d i l u t i o n o f fresh r e t i e u l o c y t e l y s a t e . Basal c y c l i c A M P f o r m a t i o n is s u b t r a c t e d t h r o u g h o u t . T h e e f f e c t o f G p p ( N H ) p in the p r e s e n c e o f l y s a t e (~) w a s f i t t e d b y a curve c a l c u l a t e d a c c o r d i n g to E q n . 1 using the f o l l o w i n g n u m e r i c a l values: K A = K B = 8 • 1 0 - 7 M , ~ = 1, ~ = 0 . 2 5 , [alA = 1 0 # M . L y s a t e GTP c o n c e n t r a t i o n [ A ] , w a s e s t i m a t e d by a trial-and-error m e t h o d , the o t h e r p a r a m e t e r s w e r e e s t i m a t e d f r o m c o n c e n t r a t i o n r e s p o n s e c u r v e s for g u a n y l n u c l e o t i d e s in t h e a b s e n c e o f l y s a t e (see p . 3 4 4 for e x p l a n a t i o n o f s y m b o l s ) . M e a n o f d u p l i c a t e d e t e r m i n a t i o n s in o n e e x p e r i m e n t . F o u r o t h e r e x p e r i m e n t s gave similar results. V a r i a t i o n o f the n u m e r i c a l values for the p a r a m e t e r s in e q u a t i o n ( 1 ) : K A = 0 . 3 - - 1 . 3 # M , K B = 0 . 6 - - 1 . 5 #M, ~ = 0.25--0.5c~, [A] = 2 - - 1 0 #M.

Fig. 6. A s y m m e t r i c e f f e c t s o f GTP and G p p ( N H ) p in partially sealed ghosts. I n c u b a t i o n t i m e w a s 4 m i n . Sealed cells c o n t r i b u t e d a p p r o x . 3 0 % o f t o t a l c y c l i c A M P ( c A M P ) f o r m a t i o n , o o, • e: GTP ( o ) or G p p ( M H ) p ( • ) p r e s e n t in t h e i n c u b a t i o n m e d i u m b u t n o t w i t h i n g h o s t s ~ A • •: GTP ( ~ ) or G p p ( N H ) p ( a ) p r e s e n t w i t h i n g h o s t s b u t n o t in t h e i n c u b a t i o n m e d i u m . E x p e r i m e n t a l p o i n t s are m e a n values o f f o u r d e t e r m i n a t i o n s in t w o i n d e p e n d e n t e x p e r i m e n t s . T w o o t h e r e x p e r i m e n t s using higher A T P c o n c e n t r a t i o n s y i e l d e d qualitatively similar results.

of cyclic AMP accumulation over the whole range of agonist concentrations. In the presence of constant free Mg 2÷ concentrations (1.3 mM) an increase in ATP from 50 pM to 2 mM shifted the K a value for GTP from 0.18 #M to 0.8 pM and reduced the relative stimulating effect of GTP on ~-adrenergic cyclic AMP formation. These effects can be explained by a small GTP contamination in the ATP charge that we have used (0.1--0.5 pM GTP/mM ATP). A much larger contamination has been observed by Kimura et al. [40].

Sidedness of guanylnucleotide action in ghost cells The effect of membrane resealing on the stimulating action of GTP and Gpp(NH)p was studied in partially sealed ghosts (Fig. 6). Two batches were prepared from the same reticulocyte suspension such that the sealed fraction of ghosts contained either 0.1 to 100 pM GTP or Gpp(NH)p (batch I) or no guanylnucleotides (batch II). Batch I was then incubated in the absence, batch II in the presence of guanylnucleotides (0.1 to 100 #M). ATP (50 #M) was present in sealed ghosts and in the incubation medium. Cyclic AMP formation in batch I and II after addition of 10 #M isoprenaline thus reflected the effect of GTP and Gpp(NH)p in sealed and unsealed ghosts, respectively. Intracellular GTP did not enhance cyclic AMP formation except for the

343 highest concentration where cellular GTP exceeded the cellular ATP content. Conversely, GTP increased cyclic AMP formation in the unsealed fraction of cells. The half-maximal effect was reached with 1 /~M GTP. Gpp(NH)p was almost inactive in unsealed cells, but strongly inhibited cyclic AMP formation if incorporated intracellularly. Half-maximal inhibition was reached with 1.5 pM Gpp(NH)p. The inhibition of the/3-adrenergic response by Gpp(NH)p was maintained for incubation periods of at least 15 min.

Interaction between fresh reticulocyte lysate and guanylnucleotide-induced stimulation of adenylate cyclase Closer inspection of these data suggested that the conspicuous differences between sealed and unsealed ghosts with respect to the effect of guanylnucleotides were mainly due to interference with residual endogenous GTP rather than to changes in the adenylate cyclase system during resealing. Since free GTP concentration in fresh reticulocytes is approx. 0.3 mM [41] resealed ghosts which trap about 1% of the original cytosol may contain up to 3 t~M GTP. This level of endogenous GTP is higher than the half-maximal effective concentration and could reduce the apparent stimulating effect of exogenous GTP. Apparently Gpp(NH)p is only a weak agonist in ghosts (see Fig. 5). According to Mass Law principles, competitive interaction of a full and a partial agonist at the same receptor system will result in an antagonistic action of the partial agonist [42]. As an experimental test for this interpretation of the intracellular actions of guanyinucleotides, we studied their effects in lysate-exposed unsealed ghosts. The cells were incubated in a 1 : 50 dilution of reticulocyte lysate supplem e n t e d with the standard components of solution VI including 0.2 mM ATP, 1.6 mM Mg 2÷ and GTP or Gpp(NH)p concentrations ranging from zero to 100 t~M (Fig. 5). Lysate alone enhanced maximally /3-adrenergic cyclase stimulation. Addition of GTP caused no further increase whereas Gpp(NH)p was strongly inhibitory. Cell lysate reduced the mean rate of basal cyclic AMP formation in the presence of 100 tiM Gpp(NH)p from 627 to 211 pmol/min per ml ghosts.

Fragmented membranes If the stimulating effect of fresh cell lysate in ghosts was due predominantly to GTP, lysate should also enhance cyclic AMP formation in fragmented membranes. The broken lines in Fig. 4 indicate that such stimulation was indeed observed. Since the efficiency of Gpp(NH)p in membranes exceeded that of GTP, the latter simply acted as an competitive antagonist. Hence, the concentrationresponse curves for the effect of Gpp(NH)p on basal and isoprenaline-stimulated cyclic AMP formation were shifted somewhat to the right. A similar result was obtained earlier in other preparations [43]. Discussion

The results suggest that in resealed reticulocyte ghosts the mammalian fl-recept0r-adenylate cyclase system can be studied under quasicellular conditions. By contrast, preparation of membrane fragments from intact cells seems

344

to alterate primarily those components o f the system that are involved in signal transfer from the receptor to the enzyme. The K a value for isoprenaline increases and the functional properties of guanylnucleotides or NaF are modified. On the other hand, properties of the receptor (binding of antagonists) and of the adenylate cyclase (Ca2+-dependent inhibition) do n o t change. Some of these points will be discussed below in more detail. Resealed hormone-sensitive ghosts have been prepared earlier from avian red cells [ 1 5 , 3 0 , 4 4 ] . The general usefulness of these systems as models for mammalian cells is limited by a number of disadvantages that are n o t shared by rat reticulocyte ghosts. Avian red cells will n o t reseal in the presence of agents that chelate divalent cations. The ghost cells lack an active Ca transport system. The cells have no measurable phosphodiesterase activity. Finally, it is difficult to prepare nucleated ghosts virtually free of cytosol. The coupling between agonist-receptor interaction and enzyme activation seems to be the most vulnerable function of the fl-adrenergic system in mammalian cell membranes [7,9]. Therefore, it is an important advantage of our model system that the Ka values for isoprenaline in resealed reticulocyte ghosts and in the corresponding intact cells are identical (Fig. 1). In binding experiments [45] the K d value for isoprenaline in native reticulocytes was 4 . 4 . 10 -~ M and hence, approx. 10-times the K a as measured in the present study ( 5 . 1 0 -8 M, if corrected for the content of the (+)-isomer in the racemic preparation). Preliminary experiments in our laboratory (Baer, M. and Porzig, H., unpublished results), suggest that the Ka : Kd ratios for isoprenaline and hence coupling efficiencies in intact cells and in GTP-loaded resealed ghosts are very similar. It is n o w generally accepted that the signal transfer from the H-receptor to the adenylate cyclase requires as a third component, a guanylnucleotide binding protein, the N-unit [46,47]. The differential effects of guanylnucleotides on both basal and fl-receptor-stimulated cyclic AMP formation in fragmented membranes and resealed ghosts (Figs. 4--6), suggest that the physiological interactions of the N-unit with the receptor and the enzyme moiety of the system are disturbed in fragmented membranes. The low activity of NaF in ghosts supports this view. In membranes Gpp(NH)p is usually the most p o t e n t activator of isoprenaline-dependent cyclic AMP formation [6,7,43]. This was also true for fragmented reticulocyte membranes (Fig. 4). By contrast, in reticulocyte ghosts the stimulating activity of GTP exceeded that of Gpp(NH)p by a factor of 2 or 3 even though no change in the K a values for the two nucleotides w a s observed. Consequently, Gpp(NH)p acted as a competitive partial antagonist of endogenous GTP in resealed ghosts. As shown in Fig. 5 the effect of Gpp(NH)p in the presence of lysate could indeed be described by the equation EAB : E M =

a/(1 + (1 + [B]/KB)KA/[A]) + 13/(1 + (1 + [A]/KA)KB/[B])

(1)

given in [42] for the interaction of two compounds, A and B, competing for the same receptor system. The ratio between the combined effect of GTP (A) and Gpp(NH)p (B), EAB, and the maximal effect, EM, depend on the concentrations of A and B, ([A], [B]) the apparent dissociation constants (KA, KB) , of the drug-receptor complex and their intrinsic activities (aft).

345

In some systems guanylnucleotides have been shown to decrease the affinity of fi-adrenergic receptors for agonists by almost one order of magnitude while increasing the Hill coefficient of agonist binding curves to near unity [7,48]. In none of our ghost preparations did we observe a significant GTPinduced shift of the KA value for isoprenaline that could have indicated a change in receptor affinity. However, the Hill coefficient for f~-adrenergic cyclase activation was significantly higher in resealed ghosts than in fragmented membranes even in the absence of added GTP (see Results). This effect is most likely to be the result of the presence of endogenous GTP in resealed ghosts rather than of a change in membrane properties during resealing. The u n k n o w n level of endogenous GTP in resealed ghosts makes it difficult to assess the contribution of membrane recovery to the overall increase in the rate of cyclic AMP formation associated with the process of resealing (Table II). With saturating concentrations of GTP, /3-adrenergic stimulation of unsealed and resealed ghosts resulted in similar maximal rates of cyclic AMP formation. However, a comparison on the basis of membrane protein content (approx. 7 mg/ml ghosts) suggests that in fragmented membranes the maximal rate of cyclic AMP formation in the presence of GTP reached only a b o u t 20% of the values in ghost preparations. Some of our experiments have tested properties of the ~-receptor or of the catalytic unit of the adenylate cyclase. The results suggest that these components survive the different preparation procedures much better than the coupling system. The affinity of the receptor for the ~-adrenergic antagonist propranolol remained unchanged during the transition from intact cells to fragmented membranes. Similarly, the high sensitivity of the enzyme for the inhibitory action of Ca 2÷ [31] was maintained in all preparations (Figs. 2 and 3). Even though continuing active Ca outward transport did not allow us to establish a stationary, well defined low [Ca]i in resealed ghosts, our results showed that concentrations in the submicromolar range caused half maximal inhibition of cyclic AMP formation in both fragmented membranes and resealed ghosts. Using the Ca-sensitive phosphoprotein obelin as an intracellular indicator in avian red cell ghosts, Campbell and Dormer [30] concluded that [Ca]i ranging between 1 and 10 pM inhibited cyclic AMP accumulation by more than 50%. These findings suggest that the rate of cyclic AMP formation in rat reticulocytes in vivo may be determined to a large extent by the actual cellular Ca 2÷ concentration. Regulatory interactions between Ca and cyclic AMP have been suggested to exist in a variety of/3-adrenergically innervated cells [1 ]. We did n o t find any evidence for a Ca-dependent stimulation of cyclase activity comparable to the one observed for adenylate cyclase from brain [49] and from glial t u m o r cells [33]. Acknowledgements This study was supported b y the Swiss National Science Foundation grant No. 3.598-0.75. Expert technical assistance of Miss S. Gurtner and Mr. F. Schmid is gratefully acknowledged. The stay of H.E. Makulska in Bern was financed by a stipend from the University of Bern.

346

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