Affinities of reactive vasopressin analogues for bovine antidiuretic receptor

Molecular and Cellular Endocrinology, 28 (1982) 529-541 Elsevier Scientific Publishers Ireland, Ltd.

AFFINITIES OF REACTIVE VASOPRESSIN BOVINE ANTIDIURETIC RECEPTOR

P. CRAUSE and F. FAHRENHOLZ Max-Planck-Insiitut

529

ANALOGUES

FOR

*

fiir Biophysik, Kennedy-Allee

70, D-6000 Frankfurt 70 (F.R.G.)

Received 9 June 1982; revision received 19 July 1982; accepted 20 July 1982

Plasma membranes containing one class of high-affinity binding sites for vasopressin were prepared from bovine kidney medulla by density gradient centrifugation in Percoll. The binding affinities of reactive analogues of [Arglvasopressin (AVP), deamino-dicarbaAVP ([ 1,6a-aminosuberic acid]AVP) and [2-phenylalanine]AVP to bovine antidiuretic receptor were determined. The peptide hormone analogues contained photoreactive azido or 4,4-azopentanoylamino residues or chemical reactive bromoacetylamino groups in the p position of Phe2 or Phe3. All azido compounds and the bromoacetyl derivative of AVP retained high binding affinities, which is a prerequisite for specific labelling of receptors. Keywords:

vasopressin;

antidiuretic receptor; affinity labelling; plasma membrane.

In the collecting duct of the inner medulla of the kidney, the neurohypophyseal hormone vasopressin regulates the flow of water across the epithelium. There is ample physiological and biochemical evidence to show that the binding site for vasopressin is on the basolateral cell membrane (Grantham and Burg, 1966; Iyengar et al., 1978) and that the interaction between the hormone and its receptor leads to an activation of the membrane-bound adenylate cyclase (Chase and Auerbach, 1968). In rabbit, rat and mouse the adenylate cyclase, which is sensitive to vasopressin, is contained not only in the medullary and cortical portions of collecting tubules, but also in the thick ascending limb of Henle’s loop (Morel et al., 1980). The design of @hoto)affinity labels for vasopressin receptors would be very helpful for studying many aspects of receptor structure, localization and function. For this purpose we prepared analogues of [Arglvasopressin (AVP) containing reactive groups which render’ covalent incorporation into proteins possible. These hormone analogues showed high intrin-

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0303-7207/82/0000-OOOOooo/$O2.75 0 Elsevier Scientific Publishers Ireland, Ltd.

530

P. Crause and F. Fahrenholz

sic activities and potencies to activate adenylate cyclase in membrane preparations from the inner medulla of bovine kidney (Fahrenholz et al., 1980). Furthermore they stimulated short-circuit current and osmotic water flow across the isolated frog skin (Fahrenholz et al., 1981). The present study reports the binding affinities of a variety of reactive AVP analogues to the antidiuretic receptor from bovine kidney inner medulla. In this context we discuss the suitability of these peptides for affinity labelling of vasopressin receptors in plasma membranes. We further describe a simple and reproducible method for preparation of membranes from bovine kidney inner medulla containing one class of high-affinity binding sites for vasopressin.

MATERIALS AND METHODS Membrane preparation

Plasma membranes from bovine kidney inner medulla were prepared by centrifugation in self-forming colloidal silica polyvinylpyrrolidone (Percoll) gradients. First a crude mitochondrial pellet was obtained as described by Fahrenholz et al. (1980). Then the pellet was resuspended in a suspension of 10% Percoll (relative to the suspension delivered by Pharmacia Fine Chemicals) containing 5 mM Hepes, pH 7.5, 0.25 M sucrose and 1 mM EDTA. Centrifugation was performed with a fixed-angle rotor at O’C and 25000 X g,,, for 60 min, using 50 ml tubes filled with 40 ml of the suspension. Fractions of 2 ml were collected by displacing the gradient with 60% (w/ w) sucrose pumped into the bottom of the tube. The fractions containing high specific activity of AVP-stimulated adenylate cyclase were pooled and the plasma membranes were separated from Percoll by high-speed centrifugation (200000 X g,, for 90 mm). A pellet was then obtained consisting of two layers, the Percoll forming the lower hard layer. The plasma membrane pellet was carefully separated from the Percoll pellet and resuspended in 5 mM Hepes-buffer, pH 8.0, containing 0.25 M sucrose and 1 mM EDTA at a concentration of 1 mg protein per ml. 1 ml aliquots were stored in liquid nitrogen for several months with no loss in vasopressin-binding capacity. Enzymatic

assays

Naf -K+ -ATPase was tested by the coupled enzyme assay described by Koepsell (1978). Vasopressin-sensitive adenylate cyclase was assayed as previously described (Fahrenholz et al., 1980) in the presence of a saturating AVP concentration (lo-’ M) and the values were corrected for basal activity. Succinic dehydrogenase was determined by an assay based

Reactive vasopressin analogues

531

on the reduction of 2,4-dichlorophenol-indophenol in the presence of succinate (Evans, 1978). In controls for non-specific reduction, succinate was displaced by malonate. Protein determination

Owing to the interference of Percoll with the Lowry method, a modification of the fluorescamine assay, described by van Frank (1975) for cell fractions, was used for protein determination. Samples (up to 200 ~1) were mixed with equal volumes of 4% sodium dodecylsulphate in 1.5 ml test tubes; 0.1 M borate buffer, pH 9, was added to a volume of 800 ~1. After shaking, 200 ~1 of 0.03% fluorescamine in acetone were added and the tubes were closed and vortexed immediately. At least 2 min later fluorescence was measured (excitation wavelength set at 390 nm, emission wavelength set at 475 nm). Peptides

Deamino-dicarba-AVP ([Asu’.~]AVP) derivatives were synthesized (Fahrenholz et al., forthcoming) by a method similar to that used for the analogues of AVP and [Zphenylalanine]AVP ([Phe’]AVP) modified in positions 2 and 3 respectively (Fahrenholz et al., 1980). Peptides containing the photoreactive diazirine group were obtained by the reaction of p-aminophenylalanine analogues with 4,4azopentanoylchloride, which was prepared by the method of Church and Weiss (1970); their a-amino groups were protected by acetone (Yamashiro et al., 1967) during the acylation of the p-amino groups. N”-Deaminotyrosylpeptides were prepared by acylation of their a-amino groups with N-succinimidyl 3-(4hydroxyphenyl)propionate (Rudinger and Ruegg, 1973). AVP was purchased from Bachem (Bubendorf, Switzerland) and deamino-dicarbaAVP from Chanical Dynamics (South Plainfield, NJ, U.S.A.). [3H]AVP was prepared by catalytic dehalogenation of the diiodo derivative as described by Morgat et al. (1970) and Pradelles et al. (1972). The radioactive ligand was further purified by affinity chromatography on a neurophysin-Sepharose4B column. The purity of [3H]AVP was demonstrated by thin-layer chromatography on silica plates. The UV-absorption spectrum of [3H]AVP was identical with that of unlabelled AVP. The specific activity of the labelled AVP was determined by measuring the absorption at h,, = 275 nm and by scintillation counting. The purified 3H-labelled product retained a specific radioactivity of 6.6 Ci/mmole. Its dissociation constant K, for binding to plasma membrane preparations from bovine kidney inner medulla was consistent with the value for unlabelled AVP, which was determined in separate competition experiments (deviation less than 10%). The radioactive hormone ligand

P. Crause and F. Fahrenhob

532

was diluted with an aqueous solution containing albumin and 1% ethanol.

0.1% bovine serum

[“HI A VP binding assay

Various initial concentrations of [3H]AVP were incubated with membranes at 30°C in the presence and absence of unlabelled lop5 M AVP. The volume of the binding assay was 200 ~1 with the following components in the medium: 25 mM N-2-hydroxyethylpiperazine-N’-3-propanesulphonic acid, pH 8.2, and 2 mM MgCl,. After 20 min incubation the medium was diluted by 5 ml of a cold solution (O’C) containing 25 mM Tris, pH 8.2, 2 mM MgCl, and 0.1% bovine serum albumin. Membranebound tritiated hormone was separated from free hormone by rapid filtration over cellulose acetate filters (0.22 pm). The filters were washed 2 times with 5 ml of the same medium, which was used for dilution, and were placed in 3 ml methylcellosolve. After the filters became transparent (l-3 min), 7 ml of a scintillation mixture (Aqualuma Plus, LKB) were added. Radioactivity was determined with an LKB scintillation counter. Specific binding was obtained by the difference between [3H]AVP binding in the absence and presence of 10m5M unlabelled AVP. The dissociation constant K, for binding of AVP and the membrane-specific binding capacity b,,,,, were determined by Scatchard plot analysis. Determination of apparent dissociation constants Ko,, of A VP analogues by displacement experiments

Binding studies were performed with lop8 M [3H]AVP for various concentrations of unlabelled AVP analogues under the conditions described above. The results were corrected for non-specific binding. The concentration of binding sites used in the assay (5.5 X lo-” M) was very low in comparison to the concentration of ligands. The concentration I,,5 of unlabelled peptide leading to 50% inhibition of specific [3H]AVP binding was obtained from a linear least-squares regression analysis of the log B/B, - B vs. log I plot, where B, is the specific binding of [ 3H]AVP in the absence of unlabelled peptide I, and B is the specific binding of [‘H]AVP in the presence of competitor I. The apparent dissociation constants K,,, were calculated using the following equation, assuming competitive interaction of the labelled and unlabelled peptides with homogeneous, non-cooperative binding sites: K

I 0.5 D,I

= gL1++ I

D,H

Here Z0.sis the total (= free) concentration

of unlabelled peptide I which

533

Reactive vasopressinanalogues

yields 50% inhibition of [3H]AVP binding, H, the concentration of free [ ‘H]AVP in the absence of the competitor I, H, the concentration of free [ 3H]AVP in the presence of I,,,, and K,,, the dissociation constant of AVP derived from the [3H]AVP dose-dependent binding; the same membrane preparation was used for the determination of K,,, as in the competition experiments. Determination

of antidiuretic activity

Antidiuretic activity was estimated on ethanol-anaesthesized rats according to the method of Jeffers et al. (1942) as modified by Pliska and Rychlik ( 1967).

RESULTS Preparation

of plasma membranes

containing binding sites for vasopressin

A partially purified plasma membrane fraction from the inner medulla of bovine kidney was prepared by differential centrifugation in isotonic sucrose and density gradient centrifugation in colloidal silica polyvinylpyrrolidone (Percoll). Fig. 1 shows the protein profile and distribution of enzymatic markers for the basal-lateral membrane (Na+ -K+ -ATPase and vasopressin-stimulated adenylate cyclase) and for mitochondria (succinic dehydrogenase) in the density gradient. The first protein peak at a density of 1.036 g/cm3 contains the plasma membrane fraction, which is clearly separated from mitochondria (second protein peak). The specific binding of [3H]AVP to a plasma membrane preparation under steady-state conditions is shown in Fig. 2. The binding sites displayed the same general properties as described by Hechter et al. (1978). The Scatchard plot of the [ ‘H]AVP binding data (Fig. 3) is linear, thereby demonstrating one class of high-affinity binding sites for [ 3H]AVP with a dissociation constant K, = 9 X lo-” M. The specific binding capacity b_ of the membrane fraction was 3.7 pmoles of [3H]AVP per mg of membrane protein. Non-cooperativity of the binding is indicated by the fact that the Hill coefficients are always close to 1.0 for different membrane preparations. When an excess of unlabelled AVP ( lop5 M) was added to membranes which had been equilibrated with lo-* M [3H]AVP, reversibility of the binding could be demonstrated (dissociation rate constant k_, = 0.014 min-’ at 30°C). Affinity of vasopressin analogues for the antidiuretic receptor

Table 1 lists the parent peptides and their analogues which were prepared for affinity labelling of vasopressin receptors. The modification

P. Crause and F. Fahrenhoiz

Fraction number

Fig. 1. Distribution of protein and enzymatic markers after Percoll density gradient centrifugation of the 10000 X g pellet from bovine kidney inner medulla in a self-forming gradient. The fractions were analysed for protein, density and enzymatic activities of AVP-stimulated adenymarkers for basal-lateral plasma membranes (Na + -K+-ATPase, late cyclase) and mitochondria (succinic dehydrogenase). The shape of the gradient, which is shown together with the protein distribution, was determined by refractive index measurements.

of

the

[Arglvasopressin

molecular

structure

Cys’-Tyr’-Phe3-

Gln4-A&-C’s6-Pro’-Arg’-Gly’-NH, by replacing the disulphide bridge by an ethylene bridge and the a-amino group by hydrogen yields deamino-dicarba-AVP ([ 1.6cY-aminosuberic acid]AVP) (2a). Replacement of Tyr’ in the AVP sequence by Phe leads to [Phe2]AVP (3a). The reactive derivatives of AVP (la) and of the compounds 2a and 3a were prepared via the precursors lb, 2b, 3b containing p-aminophenylalanine in either position 2 or 3. Modifications of the p-amino group resulted in: Azido compounds lc, 2c, 3d; Diazirine derivatives Id, 2d, 3d containing the p-4,4-azopentanoylamino residue

535

Reactive vasopressin analogues

Fig. 2. Binding of [ ‘H]AVP to bovine renal medullary membranes. The membrane preparation (30 pg per test) was incubated at 30°C for 20 min with various initial concentrations of [ 3H]AVP in the presence and absence of unlabelled AVP (low5 M). Specific binding were determined as described in 0) and non-specific binding (O- -----O) (.Materials and Methods.

Table 1 Parent peptides [8arginine]vasopressin alanine]AVP (3a) and their analogues tors

(AVP) (la), deamino-dicarba-AVP (2a), [2-phenylprepared for affinity labelling of vasopressin recep-

No.

Peptide

la lb Id le If

AVP [( p-Amino)Phe’ ]AVP [( p-Azido)Phe*JAVP [( p-4,4-Azopentanoylamino)Phe* ]AVP [(p-Bromoacetylamino)Phe*]AVP [N-(Deaminotyrosyl)-(p-azido)Phe*]AVP

2a 2b 2c 2d 2e

Deamino-dicarba-AVP [( p-Amino)Phe*]deamino-dicarba-AVP [( p-Azido)Phe*]deamino-dicarba-AVP [( p-4,4-Azopentanoylamino)Phe*]deamino-dicarba-AVP [( p-Bromoacety1amino)Phe*]deamino-dicarba-AVP

3a 3b 3c 3d 3e 3f

[Phe*]AVP [Phe*,( p-amino)Phe3]AVP [Phe*,( p-azido)Phe3]AVP [Phe*,( p-4,4-azopentanoylamino)Phe3]AVP ]Phe*,(p-bromoacetylamino)Phe”]AVP [ N”-(Deaminotyrosyl)Phe*,( p-azido)Phe’]AVP

IC

P. Crawe and F. Fahrenholz

536

N=

- [CH,],-CO-NHCH, - CY Bromoacetyl derivatives le, 2e, 3e. The azido and diazirene analogues can be converted by photoactivation into highly reactive nitrenes and carbenes respectively (Bayley and Knowles, 1978); the bromoacetyl analogues react preferentially with 0.6 CL $ T 0.4 ‘\., s !

0

1 2 3 [?-I] AVP bound (pmol/mg protein)

Fig. 3. Scatchard

analysis

4

of the binding

data in Fig. 2.

sulphydryl groups. The p-azido phenylalanine analogues lc and 3c were converted into their N-deaminotyrosyl derivatives If and 3f. These compounds can be labelled by the introduction of “‘1 (Bolton and Hunter, 1973). The relative potencies of these peptides to inhibit the specific binding

-9

-6

-7

-6

-5

-z

Peptide (log Ml

Fig. 4. Relative potencies of AVP analogues modified in position 2 for inhibiting [3H]AVP binding. A fixed concentration of [ ‘H]AVP (lo-* M) was incubated for 20 mm with a membrane preparation in the presence of unlabelled AVP analogues. The concentration of [3H]AVP used occupied about 90% of the total specific sites in the membrane (in the absence of unlabelled peptide). Non-specific binding represented 3.7% of total binding in the absence of competitor. The reduction in binding of [3H]AVP produced in 2 identical experiments by various concentrations of unlabelled peptides is shown.

Reactive

537

vasopressin analogues

-9

,

,

,

,

(

,

-6

-1

-6

-5

-4

-3

Peptide

( log M

1

Fig. 5. Relative potencies of deamino-dicarba-AVP for inhibiting [ 3H]AVP binding.

Table 2 Apparent Peptide

dissociation

constants

in position

2

KD

Ku (M)

(A’3

Id le If

9.0x10-to= 2.7X 1O-9 6.5 X 1O-9 4.4x 10-s 6.4X lo-* b 2.0x 10-7

1.0 3.0 7.2 4900 71 220

2a 2b 2c 2d 2e

2.3 X 2.5 X 5.5 x 1.7x 8.8X

1O-9 IO-* 10-s 10-s 10-s b

2.6 28 6.1 1900 9800

3c 3d 3e 3f

3.4x 2.9X 3.7x 7.5 x

10-s lo-’ 10-s b 10-7

IC

modified

K,

KD

la AVP lb

and its analogues

38 32000 41000 830

K, values of parent peptides and their analogues (with substitutions in the p position of Phe* or Phe3) for binding to the antidiuretic receptor in bovine renal medullary membranes were determined from binding and competition curves as described in Materials and Methods. a The K, value for AVP obtained from the Scatchard plot analysis of the binding data for [‘H]AVP (Fig. 3) was consistent with that of unlabelled AVP determined by separate competition experiments (deviation less than 10%). b K, values of bromoacetyl derivatives are uncorrected for possible irreversible binding.

P. Crause and F. Fahrenholz

538

-9

-0

-i

-6

-5

-L

-3

Peptide (Log Ml

Fig. 6. Relative potencies of [2-phenylalanine]AVP inhibiting [ 3H]AVP binding.

analogues

modified

in position

3 for

of [3H]AVP to plasma membranes are displayed in Figs. 4-6; their apparent dissociation constants K, for binding to the antidiuretic receptor are given in Table 2. The replacement of the phenolic hydroxyl group in position 2 of AVP by an amino or azido group yields the compounds lb and lc whose affinities remain relatively high (the factor of 3 and 7 notwithstanding). On the other hand, the introduction of the bromoacetylamino residue (le) leads to a 70-fold reduction of the binding affinity, while the introduction of the 4,4azopentanoylamino residue (Id) results in a dramatic decrease of binding affinity by a factor of 4900. In the case of deamino-dicarba-AVP (2a), the parent peptide of the second series of analogues, which differs from AVP by the replacement of the disulphide bridge by an ethylene linkage and of the o-amino group by hydrogen, a high binding affinity is found. The azido analogue in this series (2~) has about the same affinity as the corresponding azido derivative of AVP (1~); in contrast, the p-amino derivative (2b) and Table 3 Antidiuretic

activities of AVP and its analogues in anaesthetized

Peptide

rats

Antidiuretic

activity

(fU/mg) AVP (la) [( p-Azido)Phe* ]AVP (lc) Deamino-dicarba-AVP (2a) [( p-Amino)Phe*]deaminodicarba-AVP * Meienhofer et al. (1970). b Hase et al. (1972).

(2b)

503 f53a 220 2 8.5 1274 “67b 60.2k21.3

Reactive vasopressin analogues

539

particularly the p-bromoacetylamino (2e) analogue of 1-deamino-dicarbaAVP show markedly lower affinities than the counterparts derived from AVP. The derivatives of [2-phenylalanine]AVP modified in position 3 (3c-3e) have lower binding affinities than the analogues modified in position 2. The peptides with high binding affinities exhibit high intrinsic activities in stimulating adenylate cyclase (Fahrenholz et al., 1980). Their agonistic properties at the receptor level are also observed in vivo (Table 3).

DISCUSSION The combination of differential and density gradient centrifugation in colloidal silica polyvinylpyrrolidone (Percoll) is a simple and reproducible method of preparing plasma membranes from bovine kidney inner medulla. Percoll density gradient centrifugation has also been used to isolate basolateral plasma membranes from rat kidney cortex (Scalera et al., 1981). The membranes from bovine kidney inner medulla contain one class of high-affinity non-cooperative binding sites which resemble those prepared by Nakahara et al. (1978) using a different technique. In order to clarify the relationship between the structure and the binding affinity of the analogues substituted in the p position of 2-tyrosine in AVP, it would be desirable to distinguish between the effects of electronic, hydrophobic and steric properties associated with the introduction of aromatic substituents. The Hammet constant up is a measure of electronic effects and the Hansch lipophilicity constant II of the hydrophobic character (Hansch et al., 1973). Despite the replacement of the phenolic hydroxyl group (up = -0.37, II = -0.67) by the stronger electron-donating and more hydrophilic amino group (up = -0.66, II = - 1.23) or by the electron-withdrawing and lipophilic azido group (or = +0.15, II = +0.46), the high binding affinity is retained. It would thus seem that the electronic and hydrophobic properties of the substituent in the p position of residue.2 in AVP have only a weak influence on binding. The considerable decrease in the binding affinity consequent to the introduction of the bromoacetylamino group or the 4,4azopentanoylamino residue must be ascribed to the great bulk of these residues. The different binding affinities of AVP analogues and of the corresponding deamino-dicarba-AVP derivatives may reflect variations in the environment of the amino acid in position 2. Spectroscopic investigations have uncovered such differences in the backbone configurations in the

540

P. Crause and F. Fahrenholz

region of Tyr’ in oxytocin and its carba analogues (Fric et al., 1974; Kawano et al., 1979). For the specific labelling of the vasopressin receptor, we require among other things a high binding affinity and a high specific radioactivity of the reactive AVP analogue. Furthermore, the reactive group has to be in the immediate vicinity of the receptor molecule and the irreversible reaction between receptor and radioactive ligand must proceed with such a high yield that labelled proteins can be detected by analytical methods. The requirement of a high binding affinity is satisfied by all azido compounds and the bromoacetyl derivative of AVP. Owing to the low affinity of analogues containing the diazirine group, a high concentration of peptide is needed to saturate the receptor. This leads to a high degree of unspecific binding, which in turn impedes specific labelling of the receptor. The N”-deaminotyrosyl derivatives which can be labelled by 1251 with a high specific activity have a reduced binding affinity as compared to the analogues with free cY-amino groups. Tritium can be introduced as label into the reactive AVP analogues by iodination of the p-aminophenylalanine derivatives followed by catalytical exchange of iodine against tritium (Eberle and Schwyzer, 1976). Unlike neurohypophyseal hormones containing a disulphide bridge, the dicarba analogues of vasopressin can be labelled by tritium with fewer side reactions. The affinity labelling of vasopressin receptors using the tritium-labelled pazidophenylalanine derivative of deamino-dicarba-vasopressin, the derivative with the highest binding affinity in this series of analogues, is presently under investigation.

ACKNOWLEDGEMENTS The authors wish to thank Dr. T. Barth from the Institute of Organic Chemistry and Biochemistry of the Czechoslovak Academy of Science, Prague, for performing the antidiuretic tests, Dr. J.-L. Morgat from Centre d’Etudes Nucleaires de Saclay for the catalytic dehalogenation of iodinated’peptides in the presence of tritium, and Dr. K.-H. Thierauch for the neurophysin-Sepharose column. We greatfully acknowledge financial support by the Deutsche Forschungsgemeinschaft.

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541

Chase, L.R. and Aurbach, G.D. (1968) Science 159, 545. Church, R.F. and Weiss, M.J. (1970) J. Org. Chem. 35, 2465-2471. Eberle, A. and Schwyzer, R. (1976) Helv. Chim. Acta 59, 2421-2431. Evans, W.H. (1978) In: Preparation and Characterization of Mammalian Plasma Membranes (Elsevier/North-Holland, Amsterdam) pp. 118- 119. Fahrenholz, F., Thierauch, K.H. and Crause, P. (1980) Hoppe-Seyler’s Z. Physiol. Chem. 361, 153-167. Fahrenholz, F., Thierauch, K.H., Crause, P. and Johnson, A. (1981) In: Structure and Activity of Natural Peptides, Eds.: W. Voelter and G. Weitzel (Walter de Gruyter, Berlin) pp. 349-362. Fric, I., Kodicek, M., Jost, K. and Blaha, K. (1974) Coll. Czech. Chem. Commun. 39, 1271-1289. Grantham, J.J. and Burg, M.B. (1966) Am. J. Physiol. 211, 255-259. Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D. and Lien, E.J. (1973) J. Med. Chem. 16, 1207-1216. Hase, S., Sakakibara, M., Wahrunberg, M., Kirchberger, M., Schwartz, J.L. and Walter, R. (1972) J. Am. Chem. Sot. 94, 3590-3600. Hechter, O., Terada, S., Nakahara, T. and Flouret, G. (1978) J. Biol. Chem. 253,3219-3229. Iyengar, R., Mailman, D.S. and Sachs, G. (1978) Am. J. Physiol. 234, F247-F254. Jeffers, W.A., Livezey, M.M. and Austin, J.H. (1942) Proc. Sot. Exp. Biol. Med. 50, 184-188. Kawano, K., Kobayashi, Y., Kyogoku, Y., Morikawa, T. and Sakakibara,S. (1979) B&hem. Biophys. Acta 578, 87-95. Koepsell, H. (1978) J. Membrane Biol. 44, 85-102. Meienhofer, J., Trzeciak, A., Hovran, R.T. and Walter, R. (1970) J. Am. Chem. Sot. 92, 7199-7202. Morel, F., Imbert-Teboul, M. and Chabard&., D. (1980) Adv. Cyclic Nucleotide Res. 12, 301-313. Morgat, J.L., Hung, L.T., Cardinaud, R., Fromageot, P., Bockaert, J., Imbert, M. and Morel, F. (1970) J. Labelled Compounds 3, 276-284. Nakahara, T., Terada, S., Pincus, J., Flouret, G. and Hechter, 0. (1978) J. Biol. Chem. 253, 321 l-3218. Pliska, V. and Rychlik, J. (1967) Acta Endccrinol. (Kbh.) 54, 129-140. Pradelles, P., Morgat, J.L., Fromageot, P., Camier, M., Bonne, D., Cohen, P., Bockaert, J. and Jard, S. (1972) FEBS Lett. 26, 189-192. Rudinger, J. and Ruegg, U. (1973) B&hem. J. 133,538-539. Scalera, V., Huang, Y.K., Hildmann, B. and Murer, H. (1981) Membrane B&hem. 4, 49-61. Van Frank, R.M. (1975) Anal. B&hem. 65, 552-555. Yamashiro, D., Havran, R.T., Aarming, H.L. and du Vigneaud, V. (1967) Biochemistry 57, 1058-1061.