Molecular and Cellular Endocrinology
2 (1975) 69-79. 0 North-Holland
Publ. Comp.
RENAL ADENYLATE CYCLASE ACTIVATION BY AMINO ACYLATED VASOPRESSIN AND OXYTOCIN” Tomislav
BARTH,
Christian
ROY, Rabary
RAJERISON
and Serge JARD
Laboratoire de Physiologic Cellulaive, Coi@ge de France, 75231, Paris, Cedex 05, France and Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 16610, Prague 6, Czechoslovakia
Accepted
Received 21 June 1974
18 October
1974
Two series of neurohypophysial peptide amino-acylated derivatives were tested for their ability to activate plasma membrane adenylate cyclase prepared from pig or rat kidney. They were firstly [%lysine]-vasopressin-related derivatives (Na-[Glycyl-Cys]1-[8-Lysine]-vasopressin and Na-[Glycyl-Glycyl-GlycyI-Cys]‘-[8-Lysine]-vasopressin) and secondly oxytocin-related derivatives (Na-[Glycyl-Cys]l-oxytocin, Na-[Leucyl-Glycyl-Glycyl-Cys]‘-oxytocin, and Na[Glycyl-Cys]‘-[2-O-methyl tyrosinel-oxytocin). The maximal adenylate cyclase activation induced by these peptides was lower than that induced by their respective parent hormones. After incubation of these analogues with plasma membranes obtained from the renal medulla, no significant release of parent hormones occurred. Good qualitative correlations were observed between relative antidiuretic activities measured in vivo and relative potencies in activating adenylate cyclase. It was concluded that direct action of the peptides tested on the kidney is at least partly responsible for their antidiuretic activity in vivo. Keywords: amino-acylated neurohypophysial diuretic activity.
peptides;
renal adenylate
cyclase; anti-
Addition of an amino acid or a short peptide residue at the primary amino end of the oxytocin and vasopressin molecules considerably reduces their biological activities (du Vigneaud et al., 1960: Guttman and Berde, 1962; JoSt et al., 1963; BerankovCKsandrova et al., 1964). Such analogues elicit protracted effects on several biological tests. This observation together with the fact that * This work was supported by the Centre National de la Recherche Scientifique, R.C.P. grant 220 and grant 73.3 1206 from the Delegation Generale a la Recherche Scientifique et Technique (France). ** Recipient of fellowship granted by the Delegation G&&ale a la Recherche Scientifique et Technique (France).
70
T. Barth et al.
aminopeptidases present in tissue homogenates are able to release the parent hormone from the lengthened peptides led to the conclusion that such derivatives behave like ‘oxytocinogens’ and ‘vasopressinogens’ (BerBnkovb-Ksandrov& et al., 1964; Berhnkovsi-Ksandrova et al., 1966). Furthermore N-acetylated derivatives which cannot be split by aminopeptidases showed very little activity (Berde and Boissonnas, 1968). However, when tested for their antidiuretic activity in the rat, amino-acylated derivatives of oxytocin and vasopressin elicited an immediate response, which preceeded to protracted response (Rychlik, 1964). This observation suggests that these peptides are also able to interact directly with renal vasopressin receptors. It is now well established that neurohypophysial peptides exert their antidiuretic activity through an enhanced production of 3’5’-cyclic AMP (Brown et al., 1963; Edelman et al., 1964; Grantham and Burg, 1966; Grantham and Orloff, 1968). Antidiuretic hormone and its derivatives are able to induce a dose-dependent activation of the adenylate cyclase present in a membrane fraction from the kidney of several mammalian species (Dousa et al., 1971; Campbell et al., 1972; Bockaert et al., 1973a, b; Rajerison et al., 1974; Roy et al., 1974a, b). Furthermore, for a large series of vasopressin analogues, a good qualitative correlation was demonstrated between their relative antidiuretic potencies in vivo and their relative abilities to activate renal adenylate cyclase (Dousa et al., 1971; Roy et al., 1974a, b). At present, the renal adenylate cyclase activation test appears to be the best system for determining the relative abilities of neurohypophysial peptides to interact with the renal receptor involved in the antidiuretic response and for measuring their intrinsic activities on that receptor. In the present experiments, oxytocin and vasopressin aminoacylated derivatives were tested for their ability to activate pig and rat kidney adenylate cyclases, and were all found to be active. It was not possible to demonstrate a release of the parent hormones during incubation with membranes. It is thus concluded that the lengthened vasopressin and oxytocin molecules possess an intrinsic activity on the kidney.
EXPERIMENTAL
PROCEDURE
Materials
Oxytocin (oxytocin chlorhydrate) and [8-Lysinel-vasopressin ([Lysa]-vasopressin chlorhydrate) were generously donated by Dr. Boissonnas of Sandoz Ltd., Basel. Na-glycyl-[Lysa]-vasopressin (Na[Gly-Cys]‘-[Lys*]-vasopressin (Zaoral and Sorm, 1965) and N”-glycyl-glycyl-glycyl-[Lys8]-vasopressin (N”[Gly-Gly-Gly-Cys]1-[Lys8]-vasopressin (Kasafirek et al., 1966) were kindly
Renal adenylate
donated
cyclase activation
by Dr.
Kasafirek”.
by neurohypophysialpeptide
N”-glycyloxytocin
derivatives
(N”-[Gly-Cys]l-oxytocin
71
(du
Vigneaud et al., 1960; JoSt et al., 1963) and Na-Leucyl-glycyl-glycyl-glycyloxytocin (Na-[Leu-Gly-Gly-Gly-Cys]l-oxytocin (Jest et al., 1963) and Naglycyl-2-O-methyl-tyrosine-oxytocin (Na-[Gly-Cy~]1-[2-O-methyl-tyrosine]-oxytocin) were a generous gift from Dr. JoSt * *. In all cases the synthetic procedures used excluded the presence of the natural parent hormone in the preparations tested. Tris, EDTA and ATP (disodium salt) were purchased from Sigma Corp. Creatine kinase and phosphocreatine were from C. F. Boehringer, Mannheim, F. R. G. Neutral aluminium oxide (activity grade 1, batch 183) was purchased from M. Woelm, Eschwege, F.R.G. Radiochemicals [cx-~~P]-ATP was purchased from the Commissariat a 1’Energie Atomique (Saclay, France). It was stored at -20 “C in ethanol/water (SO/SO). Enzyme preparation Vasopressin-sensitive pig kidney adenylate cyclase was prepared as previously described (Bockaert et al., 1973a, b). After dissection of the kidney medulla, the tissue was homogenized in an isotonic medium kept at 4 “C (250 mM sucrose; 10 mM Tris-HCl, pH 8 ; 3.3 mM MgCl, ; 1 mM EDTA-Tris, pH 8). The 600 g pellet was then washed 5 to 6 times in the same medium rendered hypotonic by omitting the sucrose. The final pellet, referred to as the enzyme, was kept in liquid nitrogen without any loss of activity. Rat enzyme was prepared using the same procedure (Rajerison et al., 1974) except that washing was reduced to 3 times after homogenization. Rat renal adenylate cyclase was assayed within an hour after preparation since it was observed that a significant loss of activity occurs during storage (60 % loss after 24 h storage in liquid nitrogen (Rajerison et al., 1974). Adenylate cyclase activity was measured by conversion of [a- 3‘PI-ATP into cyclic [ 32P]AMP* * * as already described (Bockaert et al., 1972; Bockaert et al., 1973a b; Rajerison et al., 1974). Pig kidney adenylate cyclase activation was determined as follows: enzyme (190 to 210 ug of protein) was preincubated for 15 min at 30 “C. The incubation medium (95 ~1) contained 100 mM Tris-HCl, pH 8; 1 mM MgCl,; 0.25 mM
* Research Institute for Pharmacy and Biochemistry, Prague, Czechoslovakia. ** JoSt, K., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 16610 Prague 6, Czechoslovakia (manuscript in preparation). *** 3’5’-cyclic adenosine monophosphate.
12
T. Barth et al.
ATP; 1 mM cyclic AMP; 20 mM phosphocreatine and 100 ug creatine kinase. 0.8 pCi labelled ATP (10 ~1) was added to the preincubated samples and the reaction allowed to proceed for 5 min. It was previously controlled (Bockaert et al., 1972) that the presence of cyclic AMP (1 mM) in the incubation medium was sufficient to completely abolish the hydrolysis of the cyclic [32P]AMP formed. The preincubation period in the presence of hormone was found necessary to obtain full enzyme activation, especially for the lowest hormone concentrations (Bockaert et al., 1973a, b). When rat kidney enzyme was used the preincubation period was omitted in order to avoid the rapid loss of activity during incubation at 37 “C. The reaction was initiated by adding enzyme and allowed to proceed for 10 min. All other experimetal conditions were the same as with pig kidney enzyme except that the MgCl, concentration was 0.8 mM and the pH 7.4. The reaction was stopped by cooling and dilution of labelled precursor with an excess of unlabelled ATP (Bockaert et al., 1972). Radioactive cyclic AMP was separated by filtration on dry aluminium oxide columns (Bockaert et al., 1972) and counted by liquid scintillation in Bray’s solution (Bray, 1960). Proteins were determined according to Lowry’s method (Lowry et al., 1951) using bovine serum albumin as a standard. Adenylate cyclase activities were expressed in pmoles cyclic AMP formed per mg of protein during a lo-min period for rat enzyme, or a 5-min period for pig enzyme.
RESULTS The dose-response curves of pig and rat medullary adenylate cyclases stimulated by [8-lysinel-vasopressin, oxytocin and their amino-acylated derivatives are shown in figs. 1 (pig enzyme) and 2 (rat enzyme). A comparison of the respective responses of rat and pig enzymes to natural hormones (oxytocin and vasopressin) clearly shows that vasopressin was more active than oxytocin in both systems: the A,, values for oxytocin were very similar (for a given peptide, the A 50 value is defined as the concentration needed to obtain half the maximum activation elicited by that peptide). As reported by several authors (Dousa et al., 1971; Barth et al., 1974) pig kidney enzyme was more sensitive to [S-lysinel-vasopressin (A, D: 6.0 x 10Vg M) than rat enzyme (A,,: 2.2 x 1O-8 M). In both systems vasopressins induced higher maximal stimulations than oxytocin. In addition the shapes of the dose-response curves for the two peptides differed. In the case of oxytocin, the ratio of maximum/ threshold doses was close to 100, while for [S-lysinel-vasopressin (especially with
Renal adenyiafe cyclase acfivafion by neurohypophysial pep fide derivafives
r+4
/“‘““I
’ “““‘I
7 11”1”16‘l”“”
“‘1”“1
I ‘L’l’,’ 1 “i”“’ a ’ _
“11”“1
Fig. 1. Pig kidney adenylate cyclase activation: effects of oxytocin, [Ly?]-vasopressin and some of their amino-acylated derivatives. Pig renal plasma membranes were preincubated at 30 “C for 15 min in the presence of the indicated concentrations of peptides (tota volume 9.5 ~1). At the end of the preincuba~ion period, 10 ~1 of [G~‘PJATP was added to each sample. The adenylate cyclase activity was measured for a 5-min period at 30 “C. At the end of the incubation, reaction was stopped by diluting the labelled ATP by a large excess of unfabelied ATP. Cyclic [3ZP]AMP was separated from its labelled precursor as indicated under Methods. [Lys8J-vasopressin and oxytocin were used as reference compounds for their respective amino-acylated derivatives. Each value is the mean of two separate determinations.
the pig enzyme), dose-dependent enzyme activation was observed within a much wider concentration range (IO-lo M to lo-” M: fig. 2). The vasopressin analogues: Na-[Gly-Cys]1-[Lys8]-vasopres~in and Na[Gly-Gly-Gly-Cys]l-[Lys8]-vasoprcssin are proved to be potent activators of rat and pig kidney adenylate cyclases. Both exhibited ASo values for adenylate cyclase activation lower than that of oxytocin but signi~cantly higher than that of [Lys*]-vasopressin. Maximal activation was significantly smaller than for the parent peptide with pig kidney adenylate cyclase and even more with rat kidney adenylate cyclase. The decrease in potency and maximum activity were more pronounced for N*-[Giy-Gly-Gly-CysllfLys*]-vasopressin than
Fig. 2. Rat kidney adenylate cyclase activation: effects of oxytocin, [Ly?]-vasopressin and some of their amino-acylated derivatives. Rat renal plasma membranes were added to the incubation mixture containing the indicated concentrations of peptides and labelled ATP. Adenylate cyclase assay was performed at 37 “C for 10 min. Reaction was stopped by adding a large excess of unlabelled ATP. All subsequent steps were identical to those described under Methods. Each value is the mean of two separate determinations.
for N”-[Gly-Cys]‘-[Lys8]-vasopressin. All the amino-acylated derivatives of oxytocin behaved like partial agonists and were less potent than oxytocin in activating renal adenylate cyclases. In the pig system the oxytocin derivatives exhibited self-inhibitory properties resembling those of oxytocin but much more pronounced. Significant lowering of adenylate cyclase activity below the basal value was observed for Na-[LeuGly-Gly-Cys]l-oxytocin at 1O-3 M. In the rat system Na-[Leu-Gly-Gly-Cys]loxytocin only had self-inhibitory properties. To check whether or not the parent hormone was gradually released from lengthened peptides during incubation with kidney membranes, Na-[Gly-Cys]loxytocin (5 x lo-’ M), Na-[Gly-Cys]‘-2-O-methyl-tyrosinel-oxytocin (5 x 1O-5 M) and oxytocin (5 x 1O-5 M) were incubated for periods ranging from 1 to 60 min in the presence of rat kidney medulla membranes. At the end of the
Renal adenylate cyclase activation by neuvohypophysial peptide derivatives
75
incubation period the medium was separated by centrifugation and assayed for its ability to activate fresh rat kidney adenylate cyclase. Due to the differences in apparent A,, values observed for oxytocin and tested analogues (fig. 2) a time-dependent increase in activity would have been expected. As shown in table 1, this was not so. Incidentally, these experiments indicated that rat kidney enzyme does not inactivate oxytocin to any significant extent. In table 2, the relative activities of amino-acylated derivatives of oxytocin and vasopressin on renal adenylate cyclase are listed and compared to their relative antidiuretic activities measured in the rat. There was a fairly good correlation between these two sets of data.
Table 1 Metabolic
stability of amino-acylated derivatives of oxytocin incubated kidney medulla membranes.
Preincubation time with rat enzyme (min)
0
1 2 4 7 10 20 40 60
in presence of rat
Residual activity on rat kidney enzyme Adenylate cyclase activation (stimulated/basal) Oxytocin
Na- [Gly-Cys]‘oxytocin
Na-[Gly-Cys]‘-[2-0methyl tyrosine] oxytocin
5.23 4.72 4.22 4.21 3.73 3.57 3.93 4.61 4.93
2.92 2.67 2.52 2.78 2.33 2.26 2.53 3.15 3.22
2.15 1.96 1.77 2.07 1.87 1.84 1.76 2.41 2.49
Rat enzyme (100 pg protein) was incubated at 37 “C for the periods of time indicated in the presence of oxytocin (5 x lo-’ M), Na-[Gly-Cys]‘-oxytocin (5 x lOA M), Na-[Gly-Cys]‘[2-O-methyl tyrosinel-oxytocin (5 x 10e5 M) or in the absence of added peptide. The incubation medium (100 r-11)contained 100 mM Tris-HCI, pH 7.4; 0.8 mM MgC&; 1 mM cyclic AMP; 0.25 mM EDTA-Tris pH 7.4 and 0.25 mM ATP. At the end of the preincubation period membranes were separated by centrifugation (3000 g, 5 min at 0 “C) and the supernatant was collected. The adenylate cyclase activity of fresh rat kidney medulla membranes was assayed in the presence of 50 pulof the preincubation medium supernatant. Experimental conditions were identical to those described under Methods. The concentration of peptide in the final incubation medium was 50% of that present at the beginning of the preincubation period in the presence of rat enzyme. Adenylate cyclase activation is expressed as an activation ratio (i.e. stimulated/basal activity). Values used to calculate activation ratios are the mean of 4 determinations.
-.--
_._
-.---
Kasafirek et al. (I 966); Kynel et al. (1966) Walter et al. (1967) Du Vigneaud et al. (1960); Jogt et al. (1963); BersinkovriKsandrova et al. (1966) Jogt et al. (1960); Berankov& KsandrovB et al. (1966) JoSt et al. (in preparation)
Waiter et al. (1967) Zaoral and Sorm (1964, 1965)
Reference for synthesis and antidiuretic activity
-_-.._----
-_
-_____
._.___I_
_._____
__----
derivatives:
-.--
0.00067 0.00085
not tested
0.0089 0.00235
0.048
1 0.077
0.034
3 0.11
3
250 15-50
34
15
91 34
56
100 72
-
-t
-
-
-
0.00077
0.00126
0.0038 0.0005
0.050
1 0.091
29
38
72 44
78
100 90
I-
i-
+ +
-
-
Adenylate cyclase activation .--_________..._...__-------Antidiuretic Rat membranes Pig membranes ---. -..----_..-.._ -_____-.---activity in SelfSelfAso L.V.P. Vm,x Aso L.V.P. Vmax -----. _----. the rat: inhibitory inhibitory Aso pepttde Units/mg ASI peptide effect effect
- -
of the adenylate cyclasc activation by oxytocin, [Lys8]-vasopressin and some of their amino-acylated pig and rat adenylate cyclase responses.
L.V.P. stands for [Lys*]-vasopressin. Vmax = the maximal increase in velocity for a given peptide is expressed as a ‘A of the maximal increase induced by [Lys*]-vasopressin. It was checked that the rate of cyclic AMP production is linear over 10 min in the case of rat enzyme (Rajerison et al., 1974) and over 20 min in the case of pig enzyme (Bockaert et af., 1973s).
Na-[Leu-Gly-Gly-Cys]‘oxytocin N”-[GIy-Cys]‘-[Z-Omethyl tyrosine] oxytocin
[Lyss]-vasopressin N”-[Gly-Cysl”-[Lys*Ivasopressin Na-[Gly-Gly-Gly-Cys]‘[Lys*]-vasopressin Oxytocin N”-[Gly-Cys]“oxytocin
Compound
-_--
Characteristics
Table 2
g 2 ;r
Renal adenylate
cyclase activation
by neurohypophysial
peptide
derivatives
77
DISCUSSION The above results provide a further confirmation of previously reported observations: 1) elimination of inactivating enzymes for oxytocin and vasopressin by successive washings of a particulate fraction (600 g pellet) from kidney medulla homogenates (Bockaert et al., 1973; Barth et al., 1974); 2) self-inhibitory properties of oxytocin and its analogues (Bockaert et al., 1974; Roy et al., 1974b); 3) existence of a dose-dependent adenylate cyclase activation by [Lys*]-vasopressin over a wide concentration range (5 to 6 orders of magnitude). Since direct measurement of hormone-receptor interaction using [3H] [Lys8]-vasopressin did not reveal an heterogeneity of the population of receptor sites as regards their affinity for the hormone (Bockaert et al., 1973 ; Roy et al., 1974a, b). These observations were taken as an evidence for the existence of a non linear relationship between receptor occupancy and enzyme activation. It is also confirmed that the membrane preparation procedure preserves the specificity towards structurally related peptides of the vasopressin receptor involved in the activation of pig and rat medullary adenylate cyclase (Dousa et al., 1971; Bockaert et al., 1973a, b; Rajerison et al., 1974; Roy et al., 1974a, b; Barth et al., 1974). The above experiments clearly show that amino-acylated derivatives of both the oxytocin and vasopressin series are able to activate pig and rat kidney adenylate cyclases. However, these peptides exhibited higher A,, values and lower maximal activities as compared to their respective natural parent molecules. The biological activity of those peptides on the renal adenylate cyclase can hardly be accounted for by progressive oxytocin or vasopressin release during the incubation in the presence of kidney membranes; there was no increase in the adenylate cyclase activation by oxytocin amino-acylated analogues preciously incubated in the presence of membranes for increasing periods of time (up to 60 min). Since oxytocin is not inactivated by the enzyme preparation, a gradual release of the much more potent parent molecule would have led to an increased adenylate cyclase activation. Such a mechanism is not sufficient to explain the differences observed in maximal activations induced either by amino-acylated analogues or by parent hormone. An other possibility is that the amino-acylated derivatives behave like pure competitive inhibitors of the active hormone liberated during the incubation period in the presence of membranes. In this case too, the analogues would induce a maximal response identical to that of the parent hormone when preincubated in the presence of membranes for a long period of time. The data presented in ligs. 1 and 2 and table 1 rule out this possibility. Moreover as reported by Berankova-Ksandrova
78
T. Barth et al.
al. (1966) Na-[Gly-Cys]‘-oxytocin did not inhibit the in vivo antidiuretic response to oxytocin. The most Iikely explanation of the above described resufts is that the tested amino-acylated derivatives of oxytocin and vasopressin are able to directly activate the renal medullary adenylate cyclase and consequently that at least part of their in vivo antidiuretic effect results from a direct interaction with the antidiuretic hormone renal receptors. On the other hand, the ‘protracted’ character of the antidiuret~c response to these analogues might be related to enhanced metabolic stability, larger distribution space, reduced elimination rate from the circulating blood or slower turnover of the peptide molecules on the antidiuretic hormone receptor. The observed activity on the renal adenylate cyclase of extended-chain analogues further confirms the conclusion of Roy et al. (1974a, b), that substitution or elimination of the primary amino group of cystein 1 in the oxytocin or vasopressin molecules is compatible with for such modified peptides to interact with the renal antidiuretic hormone receptor. Addition of a tripeptide chain at the amino end of vasopressin molecule in Na-[Gly-Gly-Gly-Cys]l[Lyss]-vasopressin only reduces the A 50 value by a factor of 20 when compared to [Lyss]-vasopressin (see table 2). et
ACKNOWLEDGEMENTS We are grateful to Drs. R. Jo& and E. Kasafirek for their kind supply of neurohypophysia~ peptide derivatives used in this study. The skilful technical assistance of Miss M. Bloch is warmly appreciated.
REFERENCES Barth, T., Rajerison, R., Roy, C. and Jard, S. Submitted for publication. Bednkov&Ksandrov& Z., B&set, G. W., Jogt, K., KrejCf, I., Plilka, V., Rudinger, J., Rychiik, I. and Sorm, F. (1966) Brit. J. Pharmacol. 24, 615. BerBnkova-Ksandrova, Z., Rychlik, I. and Sorm, F. (1964) In: Oxytocin, Vasopressin and their Structural Analogues, 1st ed., Vol. 10, Ed.: J. Rudinger (Pergamon Press, Oxford)
p. 181. Berde, B. and Boissonnas, R. A. (1968) in: Handbook of Experimental Pharmacology, 1st Ed., Vol. 23, Ed.: B. Berde (Springer Verlag, Berlin) p. 802. Bockaert, .I., Roy, C!., Rajerison, R. and Jard, S. (1973a) J. Biol. Chem. 248, 5922. Bockaert, J., Roy, C., Rajerison, R. and Jard, S. (1973b) Compt. Rend. Acad. Sci. Paris 276, 649. Bockaert, J., Roy, C. and Jard, S. (1972) J. Biol. Chem. 247, ‘7073.
Renal adenylate
cyclase activation
by neurohypophysial
peptide
derivatives
79
Bray, G. A. (1960) Anal. Biochem. 1, 279. Brown, E., Clarke, D. L., Roux, V. and Sherman, G. H. (1963) J. Biol. Chem. 238, pc 852. Campbell, B. J., Woodward, G., and Borberg, V. (1972) J. Biol. Chem. 247, 6167. Dousa, T., Hechter, O., Schwartz, I. L. and Walter, R. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1693. Edelman, I. S., Petersen, M. J. and Gulyassy, P. F. (1964) J. Clin. Invest. 43, 2185. Grantham, J. J. and Burg, M. G. (1966) Amer. J. Physiol. 211, 255. Grantham, J. and Orloff, J. (1968) J. Clin. Invest. 47, 1154. Guttmann, S. and Berde, B. (1962) Helvetica Chim. Acta 45, 2517. Jo%, K., Rudinger, J. and sorm, F. (1963) Coil. Czech. Chem. Commun. 28, 2021. Kasafirek, E., Rgbek, V., Rudinger, J. and Sorm, F. (1966) Coil. Czech. Chem. Commun. 31, 4581. Kynel, J., Plibka, V. and Jelinek, V. (1966) Desk. Fysiol. 15, 398. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265. Rajerison, R., Marchetti, J., Roy, C., Bockaert, J. and Jard, S. (1974) J. Biol. Chem. (in press). Roy, C., Barth, T. and Jard, S. (1974a) J. Biol. Chem. (accepted for publication). Roy, C., Barth, T. and Jard, S. (1974b) J. Biol. Chem. (accepted for publication). Rychlik, I. (1964) In: Oxytocin, Vasopressin and their Structural Analogues, 1st Ed., Vol. 10, Ed.: J. Rudinger (Pargamon Press, Oxford) p. 153. Vigneaud, V. du, Fitt, P. S., Bodansky, M. and O’Connell, M. (1960) Proc. Sot. Exptl. Biol. Med. 104, 653. Walter, R., Rudinger, J. and Schwartz, I. L. (1967) Amer. J. Med. 42, 653. Zaoral, M. and sorm, F. (1964) In: Oxytocin, Vasopressin and their Structural Analogues, 1st Ed., Vol. 10, Ed.: J. Rudinger (Pergamon Press, Oxford) p. 167. Zaoral, M. and sorm, F. (1965) Coil. Czech. Chem. Commun. 30,2812.
2 (1975) 69-79. 0 North-Holland
Publ. Comp.
RENAL ADENYLATE CYCLASE ACTIVATION BY AMINO ACYLATED VASOPRESSIN AND OXYTOCIN” Tomislav
BARTH,
Christian
ROY, Rabary
RAJERISON
and Serge JARD
Laboratoire de Physiologic Cellulaive, Coi@ge de France, 75231, Paris, Cedex 05, France and Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 16610, Prague 6, Czechoslovakia
Accepted
Received 21 June 1974
18 October
1974
Two series of neurohypophysial peptide amino-acylated derivatives were tested for their ability to activate plasma membrane adenylate cyclase prepared from pig or rat kidney. They were firstly [%lysine]-vasopressin-related derivatives (Na-[Glycyl-Cys]1-[8-Lysine]-vasopressin and Na-[Glycyl-Glycyl-GlycyI-Cys]‘-[8-Lysine]-vasopressin) and secondly oxytocin-related derivatives (Na-[Glycyl-Cys]l-oxytocin, Na-[Leucyl-Glycyl-Glycyl-Cys]‘-oxytocin, and Na[Glycyl-Cys]‘-[2-O-methyl tyrosinel-oxytocin). The maximal adenylate cyclase activation induced by these peptides was lower than that induced by their respective parent hormones. After incubation of these analogues with plasma membranes obtained from the renal medulla, no significant release of parent hormones occurred. Good qualitative correlations were observed between relative antidiuretic activities measured in vivo and relative potencies in activating adenylate cyclase. It was concluded that direct action of the peptides tested on the kidney is at least partly responsible for their antidiuretic activity in vivo. Keywords: amino-acylated neurohypophysial diuretic activity.
peptides;
renal adenylate
cyclase; anti-
Addition of an amino acid or a short peptide residue at the primary amino end of the oxytocin and vasopressin molecules considerably reduces their biological activities (du Vigneaud et al., 1960: Guttman and Berde, 1962; JoSt et al., 1963; BerankovCKsandrova et al., 1964). Such analogues elicit protracted effects on several biological tests. This observation together with the fact that * This work was supported by the Centre National de la Recherche Scientifique, R.C.P. grant 220 and grant 73.3 1206 from the Delegation Generale a la Recherche Scientifique et Technique (France). ** Recipient of fellowship granted by the Delegation G&&ale a la Recherche Scientifique et Technique (France).
70
T. Barth et al.
aminopeptidases present in tissue homogenates are able to release the parent hormone from the lengthened peptides led to the conclusion that such derivatives behave like ‘oxytocinogens’ and ‘vasopressinogens’ (BerBnkovb-Ksandrov& et al., 1964; Berhnkovsi-Ksandrova et al., 1966). Furthermore N-acetylated derivatives which cannot be split by aminopeptidases showed very little activity (Berde and Boissonnas, 1968). However, when tested for their antidiuretic activity in the rat, amino-acylated derivatives of oxytocin and vasopressin elicited an immediate response, which preceeded to protracted response (Rychlik, 1964). This observation suggests that these peptides are also able to interact directly with renal vasopressin receptors. It is now well established that neurohypophysial peptides exert their antidiuretic activity through an enhanced production of 3’5’-cyclic AMP (Brown et al., 1963; Edelman et al., 1964; Grantham and Burg, 1966; Grantham and Orloff, 1968). Antidiuretic hormone and its derivatives are able to induce a dose-dependent activation of the adenylate cyclase present in a membrane fraction from the kidney of several mammalian species (Dousa et al., 1971; Campbell et al., 1972; Bockaert et al., 1973a, b; Rajerison et al., 1974; Roy et al., 1974a, b). Furthermore, for a large series of vasopressin analogues, a good qualitative correlation was demonstrated between their relative antidiuretic potencies in vivo and their relative abilities to activate renal adenylate cyclase (Dousa et al., 1971; Roy et al., 1974a, b). At present, the renal adenylate cyclase activation test appears to be the best system for determining the relative abilities of neurohypophysial peptides to interact with the renal receptor involved in the antidiuretic response and for measuring their intrinsic activities on that receptor. In the present experiments, oxytocin and vasopressin aminoacylated derivatives were tested for their ability to activate pig and rat kidney adenylate cyclases, and were all found to be active. It was not possible to demonstrate a release of the parent hormones during incubation with membranes. It is thus concluded that the lengthened vasopressin and oxytocin molecules possess an intrinsic activity on the kidney.
EXPERIMENTAL
PROCEDURE
Materials
Oxytocin (oxytocin chlorhydrate) and [8-Lysinel-vasopressin ([Lysa]-vasopressin chlorhydrate) were generously donated by Dr. Boissonnas of Sandoz Ltd., Basel. Na-glycyl-[Lysa]-vasopressin (Na[Gly-Cys]‘-[Lys*]-vasopressin (Zaoral and Sorm, 1965) and N”-glycyl-glycyl-glycyl-[Lys8]-vasopressin (N”[Gly-Gly-Gly-Cys]1-[Lys8]-vasopressin (Kasafirek et al., 1966) were kindly
Renal adenylate
donated
cyclase activation
by Dr.
Kasafirek”.
by neurohypophysialpeptide
N”-glycyloxytocin
derivatives
(N”-[Gly-Cys]l-oxytocin
71
(du
Vigneaud et al., 1960; JoSt et al., 1963) and Na-Leucyl-glycyl-glycyl-glycyloxytocin (Na-[Leu-Gly-Gly-Gly-Cys]l-oxytocin (Jest et al., 1963) and Naglycyl-2-O-methyl-tyrosine-oxytocin (Na-[Gly-Cy~]1-[2-O-methyl-tyrosine]-oxytocin) were a generous gift from Dr. JoSt * *. In all cases the synthetic procedures used excluded the presence of the natural parent hormone in the preparations tested. Tris, EDTA and ATP (disodium salt) were purchased from Sigma Corp. Creatine kinase and phosphocreatine were from C. F. Boehringer, Mannheim, F. R. G. Neutral aluminium oxide (activity grade 1, batch 183) was purchased from M. Woelm, Eschwege, F.R.G. Radiochemicals [cx-~~P]-ATP was purchased from the Commissariat a 1’Energie Atomique (Saclay, France). It was stored at -20 “C in ethanol/water (SO/SO). Enzyme preparation Vasopressin-sensitive pig kidney adenylate cyclase was prepared as previously described (Bockaert et al., 1973a, b). After dissection of the kidney medulla, the tissue was homogenized in an isotonic medium kept at 4 “C (250 mM sucrose; 10 mM Tris-HCl, pH 8 ; 3.3 mM MgCl, ; 1 mM EDTA-Tris, pH 8). The 600 g pellet was then washed 5 to 6 times in the same medium rendered hypotonic by omitting the sucrose. The final pellet, referred to as the enzyme, was kept in liquid nitrogen without any loss of activity. Rat enzyme was prepared using the same procedure (Rajerison et al., 1974) except that washing was reduced to 3 times after homogenization. Rat renal adenylate cyclase was assayed within an hour after preparation since it was observed that a significant loss of activity occurs during storage (60 % loss after 24 h storage in liquid nitrogen (Rajerison et al., 1974). Adenylate cyclase activity was measured by conversion of [a- 3‘PI-ATP into cyclic [ 32P]AMP* * * as already described (Bockaert et al., 1972; Bockaert et al., 1973a b; Rajerison et al., 1974). Pig kidney adenylate cyclase activation was determined as follows: enzyme (190 to 210 ug of protein) was preincubated for 15 min at 30 “C. The incubation medium (95 ~1) contained 100 mM Tris-HCl, pH 8; 1 mM MgCl,; 0.25 mM
* Research Institute for Pharmacy and Biochemistry, Prague, Czechoslovakia. ** JoSt, K., Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 16610 Prague 6, Czechoslovakia (manuscript in preparation). *** 3’5’-cyclic adenosine monophosphate.
12
T. Barth et al.
ATP; 1 mM cyclic AMP; 20 mM phosphocreatine and 100 ug creatine kinase. 0.8 pCi labelled ATP (10 ~1) was added to the preincubated samples and the reaction allowed to proceed for 5 min. It was previously controlled (Bockaert et al., 1972) that the presence of cyclic AMP (1 mM) in the incubation medium was sufficient to completely abolish the hydrolysis of the cyclic [32P]AMP formed. The preincubation period in the presence of hormone was found necessary to obtain full enzyme activation, especially for the lowest hormone concentrations (Bockaert et al., 1973a, b). When rat kidney enzyme was used the preincubation period was omitted in order to avoid the rapid loss of activity during incubation at 37 “C. The reaction was initiated by adding enzyme and allowed to proceed for 10 min. All other experimetal conditions were the same as with pig kidney enzyme except that the MgCl, concentration was 0.8 mM and the pH 7.4. The reaction was stopped by cooling and dilution of labelled precursor with an excess of unlabelled ATP (Bockaert et al., 1972). Radioactive cyclic AMP was separated by filtration on dry aluminium oxide columns (Bockaert et al., 1972) and counted by liquid scintillation in Bray’s solution (Bray, 1960). Proteins were determined according to Lowry’s method (Lowry et al., 1951) using bovine serum albumin as a standard. Adenylate cyclase activities were expressed in pmoles cyclic AMP formed per mg of protein during a lo-min period for rat enzyme, or a 5-min period for pig enzyme.
RESULTS The dose-response curves of pig and rat medullary adenylate cyclases stimulated by [8-lysinel-vasopressin, oxytocin and their amino-acylated derivatives are shown in figs. 1 (pig enzyme) and 2 (rat enzyme). A comparison of the respective responses of rat and pig enzymes to natural hormones (oxytocin and vasopressin) clearly shows that vasopressin was more active than oxytocin in both systems: the A,, values for oxytocin were very similar (for a given peptide, the A 50 value is defined as the concentration needed to obtain half the maximum activation elicited by that peptide). As reported by several authors (Dousa et al., 1971; Barth et al., 1974) pig kidney enzyme was more sensitive to [S-lysinel-vasopressin (A, D: 6.0 x 10Vg M) than rat enzyme (A,,: 2.2 x 1O-8 M). In both systems vasopressins induced higher maximal stimulations than oxytocin. In addition the shapes of the dose-response curves for the two peptides differed. In the case of oxytocin, the ratio of maximum/ threshold doses was close to 100, while for [S-lysinel-vasopressin (especially with
Renal adenyiafe cyclase acfivafion by neurohypophysial pep fide derivafives
r+4
/“‘““I
’ “““‘I
7 11”1”16‘l”“”
“‘1”“1
I ‘L’l’,’ 1 “i”“’ a ’ _
“11”“1
Fig. 1. Pig kidney adenylate cyclase activation: effects of oxytocin, [Ly?]-vasopressin and some of their amino-acylated derivatives. Pig renal plasma membranes were preincubated at 30 “C for 15 min in the presence of the indicated concentrations of peptides (tota volume 9.5 ~1). At the end of the preincuba~ion period, 10 ~1 of [G~‘PJATP was added to each sample. The adenylate cyclase activity was measured for a 5-min period at 30 “C. At the end of the incubation, reaction was stopped by diluting the labelled ATP by a large excess of unfabelied ATP. Cyclic [3ZP]AMP was separated from its labelled precursor as indicated under Methods. [Lys8J-vasopressin and oxytocin were used as reference compounds for their respective amino-acylated derivatives. Each value is the mean of two separate determinations.
the pig enzyme), dose-dependent enzyme activation was observed within a much wider concentration range (IO-lo M to lo-” M: fig. 2). The vasopressin analogues: Na-[Gly-Cys]1-[Lys8]-vasopres~in and Na[Gly-Gly-Gly-Cys]l-[Lys8]-vasoprcssin are proved to be potent activators of rat and pig kidney adenylate cyclases. Both exhibited ASo values for adenylate cyclase activation lower than that of oxytocin but signi~cantly higher than that of [Lys*]-vasopressin. Maximal activation was significantly smaller than for the parent peptide with pig kidney adenylate cyclase and even more with rat kidney adenylate cyclase. The decrease in potency and maximum activity were more pronounced for N*-[Giy-Gly-Gly-CysllfLys*]-vasopressin than
Fig. 2. Rat kidney adenylate cyclase activation: effects of oxytocin, [Ly?]-vasopressin and some of their amino-acylated derivatives. Rat renal plasma membranes were added to the incubation mixture containing the indicated concentrations of peptides and labelled ATP. Adenylate cyclase assay was performed at 37 “C for 10 min. Reaction was stopped by adding a large excess of unlabelled ATP. All subsequent steps were identical to those described under Methods. Each value is the mean of two separate determinations.
for N”-[Gly-Cys]‘-[Lys8]-vasopressin. All the amino-acylated derivatives of oxytocin behaved like partial agonists and were less potent than oxytocin in activating renal adenylate cyclases. In the pig system the oxytocin derivatives exhibited self-inhibitory properties resembling those of oxytocin but much more pronounced. Significant lowering of adenylate cyclase activity below the basal value was observed for Na-[LeuGly-Gly-Cys]l-oxytocin at 1O-3 M. In the rat system Na-[Leu-Gly-Gly-Cys]loxytocin only had self-inhibitory properties. To check whether or not the parent hormone was gradually released from lengthened peptides during incubation with kidney membranes, Na-[Gly-Cys]loxytocin (5 x lo-’ M), Na-[Gly-Cys]‘-2-O-methyl-tyrosinel-oxytocin (5 x 1O-5 M) and oxytocin (5 x 1O-5 M) were incubated for periods ranging from 1 to 60 min in the presence of rat kidney medulla membranes. At the end of the
Renal adenylate cyclase activation by neuvohypophysial peptide derivatives
75
incubation period the medium was separated by centrifugation and assayed for its ability to activate fresh rat kidney adenylate cyclase. Due to the differences in apparent A,, values observed for oxytocin and tested analogues (fig. 2) a time-dependent increase in activity would have been expected. As shown in table 1, this was not so. Incidentally, these experiments indicated that rat kidney enzyme does not inactivate oxytocin to any significant extent. In table 2, the relative activities of amino-acylated derivatives of oxytocin and vasopressin on renal adenylate cyclase are listed and compared to their relative antidiuretic activities measured in the rat. There was a fairly good correlation between these two sets of data.
Table 1 Metabolic
stability of amino-acylated derivatives of oxytocin incubated kidney medulla membranes.
Preincubation time with rat enzyme (min)
0
1 2 4 7 10 20 40 60
in presence of rat
Residual activity on rat kidney enzyme Adenylate cyclase activation (stimulated/basal) Oxytocin
Na- [Gly-Cys]‘oxytocin
Na-[Gly-Cys]‘-[2-0methyl tyrosine] oxytocin
5.23 4.72 4.22 4.21 3.73 3.57 3.93 4.61 4.93
2.92 2.67 2.52 2.78 2.33 2.26 2.53 3.15 3.22
2.15 1.96 1.77 2.07 1.87 1.84 1.76 2.41 2.49
Rat enzyme (100 pg protein) was incubated at 37 “C for the periods of time indicated in the presence of oxytocin (5 x lo-’ M), Na-[Gly-Cys]‘-oxytocin (5 x lOA M), Na-[Gly-Cys]‘[2-O-methyl tyrosinel-oxytocin (5 x 10e5 M) or in the absence of added peptide. The incubation medium (100 r-11)contained 100 mM Tris-HCI, pH 7.4; 0.8 mM MgC&; 1 mM cyclic AMP; 0.25 mM EDTA-Tris pH 7.4 and 0.25 mM ATP. At the end of the preincubation period membranes were separated by centrifugation (3000 g, 5 min at 0 “C) and the supernatant was collected. The adenylate cyclase activity of fresh rat kidney medulla membranes was assayed in the presence of 50 pulof the preincubation medium supernatant. Experimental conditions were identical to those described under Methods. The concentration of peptide in the final incubation medium was 50% of that present at the beginning of the preincubation period in the presence of rat enzyme. Adenylate cyclase activation is expressed as an activation ratio (i.e. stimulated/basal activity). Values used to calculate activation ratios are the mean of 4 determinations.
-.--
_._
-.---
Kasafirek et al. (I 966); Kynel et al. (1966) Walter et al. (1967) Du Vigneaud et al. (1960); Jogt et al. (1963); BersinkovriKsandrova et al. (1966) Jogt et al. (1960); Berankov& KsandrovB et al. (1966) JoSt et al. (in preparation)
Waiter et al. (1967) Zaoral and Sorm (1964, 1965)
Reference for synthesis and antidiuretic activity
-_-.._----
-_
-_____
._.___I_
_._____
__----
derivatives:
-.--
0.00067 0.00085
not tested
0.0089 0.00235
0.048
1 0.077
0.034
3 0.11
3
250 15-50
34
15
91 34
56
100 72
-
-t
-
-
-
0.00077
0.00126
0.0038 0.0005
0.050
1 0.091
29
38
72 44
78
100 90
I-
i-
+ +
-
-
Adenylate cyclase activation .--_________..._...__-------Antidiuretic Rat membranes Pig membranes ---. -..----_..-.._ -_____-.---activity in SelfSelfAso L.V.P. Vm,x Aso L.V.P. Vmax -----. _----. the rat: inhibitory inhibitory Aso pepttde Units/mg ASI peptide effect effect
- -
of the adenylate cyclasc activation by oxytocin, [Lys8]-vasopressin and some of their amino-acylated pig and rat adenylate cyclase responses.
L.V.P. stands for [Lys*]-vasopressin. Vmax = the maximal increase in velocity for a given peptide is expressed as a ‘A of the maximal increase induced by [Lys*]-vasopressin. It was checked that the rate of cyclic AMP production is linear over 10 min in the case of rat enzyme (Rajerison et al., 1974) and over 20 min in the case of pig enzyme (Bockaert et af., 1973s).
Na-[Leu-Gly-Gly-Cys]‘oxytocin N”-[GIy-Cys]‘-[Z-Omethyl tyrosine] oxytocin
[Lyss]-vasopressin N”-[Gly-Cysl”-[Lys*Ivasopressin Na-[Gly-Gly-Gly-Cys]‘[Lys*]-vasopressin Oxytocin N”-[Gly-Cys]“oxytocin
Compound
-_--
Characteristics
Table 2
g 2 ;r
Renal adenylate
cyclase activation
by neurohypophysial
peptide
derivatives
77
DISCUSSION The above results provide a further confirmation of previously reported observations: 1) elimination of inactivating enzymes for oxytocin and vasopressin by successive washings of a particulate fraction (600 g pellet) from kidney medulla homogenates (Bockaert et al., 1973; Barth et al., 1974); 2) self-inhibitory properties of oxytocin and its analogues (Bockaert et al., 1974; Roy et al., 1974b); 3) existence of a dose-dependent adenylate cyclase activation by [Lys*]-vasopressin over a wide concentration range (5 to 6 orders of magnitude). Since direct measurement of hormone-receptor interaction using [3H] [Lys8]-vasopressin did not reveal an heterogeneity of the population of receptor sites as regards their affinity for the hormone (Bockaert et al., 1973 ; Roy et al., 1974a, b). These observations were taken as an evidence for the existence of a non linear relationship between receptor occupancy and enzyme activation. It is also confirmed that the membrane preparation procedure preserves the specificity towards structurally related peptides of the vasopressin receptor involved in the activation of pig and rat medullary adenylate cyclase (Dousa et al., 1971; Bockaert et al., 1973a, b; Rajerison et al., 1974; Roy et al., 1974a, b; Barth et al., 1974). The above experiments clearly show that amino-acylated derivatives of both the oxytocin and vasopressin series are able to activate pig and rat kidney adenylate cyclases. However, these peptides exhibited higher A,, values and lower maximal activities as compared to their respective natural parent molecules. The biological activity of those peptides on the renal adenylate cyclase can hardly be accounted for by progressive oxytocin or vasopressin release during the incubation in the presence of kidney membranes; there was no increase in the adenylate cyclase activation by oxytocin amino-acylated analogues preciously incubated in the presence of membranes for increasing periods of time (up to 60 min). Since oxytocin is not inactivated by the enzyme preparation, a gradual release of the much more potent parent molecule would have led to an increased adenylate cyclase activation. Such a mechanism is not sufficient to explain the differences observed in maximal activations induced either by amino-acylated analogues or by parent hormone. An other possibility is that the amino-acylated derivatives behave like pure competitive inhibitors of the active hormone liberated during the incubation period in the presence of membranes. In this case too, the analogues would induce a maximal response identical to that of the parent hormone when preincubated in the presence of membranes for a long period of time. The data presented in ligs. 1 and 2 and table 1 rule out this possibility. Moreover as reported by Berankova-Ksandrova
78
T. Barth et al.
al. (1966) Na-[Gly-Cys]‘-oxytocin did not inhibit the in vivo antidiuretic response to oxytocin. The most Iikely explanation of the above described resufts is that the tested amino-acylated derivatives of oxytocin and vasopressin are able to directly activate the renal medullary adenylate cyclase and consequently that at least part of their in vivo antidiuretic effect results from a direct interaction with the antidiuretic hormone renal receptors. On the other hand, the ‘protracted’ character of the antidiuret~c response to these analogues might be related to enhanced metabolic stability, larger distribution space, reduced elimination rate from the circulating blood or slower turnover of the peptide molecules on the antidiuretic hormone receptor. The observed activity on the renal adenylate cyclase of extended-chain analogues further confirms the conclusion of Roy et al. (1974a, b), that substitution or elimination of the primary amino group of cystein 1 in the oxytocin or vasopressin molecules is compatible with for such modified peptides to interact with the renal antidiuretic hormone receptor. Addition of a tripeptide chain at the amino end of vasopressin molecule in Na-[Gly-Gly-Gly-Cys]l[Lyss]-vasopressin only reduces the A 50 value by a factor of 20 when compared to [Lyss]-vasopressin (see table 2). et
ACKNOWLEDGEMENTS We are grateful to Drs. R. Jo& and E. Kasafirek for their kind supply of neurohypophysia~ peptide derivatives used in this study. The skilful technical assistance of Miss M. Bloch is warmly appreciated.
REFERENCES Barth, T., Rajerison, R., Roy, C. and Jard, S. Submitted for publication. Bednkov&Ksandrov& Z., B&set, G. W., Jogt, K., KrejCf, I., Plilka, V., Rudinger, J., Rychiik, I. and Sorm, F. (1966) Brit. J. Pharmacol. 24, 615. BerBnkova-Ksandrova, Z., Rychlik, I. and Sorm, F. (1964) In: Oxytocin, Vasopressin and their Structural Analogues, 1st ed., Vol. 10, Ed.: J. Rudinger (Pergamon Press, Oxford)
p. 181. Berde, B. and Boissonnas, R. A. (1968) in: Handbook of Experimental Pharmacology, 1st Ed., Vol. 23, Ed.: B. Berde (Springer Verlag, Berlin) p. 802. Bockaert, .I., Roy, C!., Rajerison, R. and Jard, S. (1973a) J. Biol. Chem. 248, 5922. Bockaert, J., Roy, C., Rajerison, R. and Jard, S. (1973b) Compt. Rend. Acad. Sci. Paris 276, 649. Bockaert, J., Roy, C. and Jard, S. (1972) J. Biol. Chem. 247, ‘7073.
Renal adenylate
cyclase activation
by neurohypophysial
peptide
derivatives
79
Bray, G. A. (1960) Anal. Biochem. 1, 279. Brown, E., Clarke, D. L., Roux, V. and Sherman, G. H. (1963) J. Biol. Chem. 238, pc 852. Campbell, B. J., Woodward, G., and Borberg, V. (1972) J. Biol. Chem. 247, 6167. Dousa, T., Hechter, O., Schwartz, I. L. and Walter, R. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1693. Edelman, I. S., Petersen, M. J. and Gulyassy, P. F. (1964) J. Clin. Invest. 43, 2185. Grantham, J. J. and Burg, M. G. (1966) Amer. J. Physiol. 211, 255. Grantham, J. and Orloff, J. (1968) J. Clin. Invest. 47, 1154. Guttmann, S. and Berde, B. (1962) Helvetica Chim. Acta 45, 2517. Jo%, K., Rudinger, J. and sorm, F. (1963) Coil. Czech. Chem. Commun. 28, 2021. Kasafirek, E., Rgbek, V., Rudinger, J. and Sorm, F. (1966) Coil. Czech. Chem. Commun. 31, 4581. Kynel, J., Plibka, V. and Jelinek, V. (1966) Desk. Fysiol. 15, 398. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265. Rajerison, R., Marchetti, J., Roy, C., Bockaert, J. and Jard, S. (1974) J. Biol. Chem. (in press). Roy, C., Barth, T. and Jard, S. (1974a) J. Biol. Chem. (accepted for publication). Roy, C., Barth, T. and Jard, S. (1974b) J. Biol. Chem. (accepted for publication). Rychlik, I. (1964) In: Oxytocin, Vasopressin and their Structural Analogues, 1st Ed., Vol. 10, Ed.: J. Rudinger (Pargamon Press, Oxford) p. 153. Vigneaud, V. du, Fitt, P. S., Bodansky, M. and O’Connell, M. (1960) Proc. Sot. Exptl. Biol. Med. 104, 653. Walter, R., Rudinger, J. and Schwartz, I. L. (1967) Amer. J. Med. 42, 653. Zaoral, M. and sorm, F. (1964) In: Oxytocin, Vasopressin and their Structural Analogues, 1st Ed., Vol. 10, Ed.: J. Rudinger (Pergamon Press, Oxford) p. 167. Zaoral, M. and sorm, F. (1965) Coil. Czech. Chem. Commun. 30,2812.
