Neurohypophyseal Hormone-Sensitive Adenyl Cyclase of Toad Urinary Bladder* - PNAS
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Proceeding8 of the National Academy of Sciences
Vol. 67, No. 1, pp. 7-12, September 1970
Neurohypophyseal Hormone- Sensitive Adenyl Cyclase of
Toad Urinary Bladder*
Hans-Peter Bar,t Oscar Hechter, t Irving L. Schwartz,
and Roderich Walter
AMERICAN MEDICAL ASSOCIATION, CHICAGO, ILLINOIS; BROOKHAVEN NATIONAL LABOLVI'OIiY,
UPTON, NEW YORK; AND THE MOUNT SINAI MEDICAL SCHOOL OF THE CITY UNIVERSITY OF
NEW YORK, NEW YORK
Communicated by Donald D. Van Slyke, June 4, 1970
Abstract. An adenyl cyclase preparation derived from epithelial cells of the
urinary bladder of the toad, Bufo marinus, is described. This cyclase prepara-
tion is specifically stimulated by neurohypophyseal hormones and various
synthetic analogs which evoke a hydroosmotic response in the intact bladder.
The relative stimulatory effects of these compounds have been compared on the
cyclase preparation and in the intact bladder. The peptide concentrations re-
quired for half-maximal stimulation (affinity) in the cell-free and intact systems
were parallel; however the magnitude of stimulation produced by saturating
concentrations of peptides did not correlate. Furthermore, it was found that
peptide analogs which inhibit the hydroosmotic effect of [8-arginine ]-vasopressin
on the intact bladder also inhibit the stimulation of the toad bladder cyclase
preparation by vasopressin. Prostaglandin E1, mercaptans, and disulfides,
which inhibit the hormone-induced hydroosmotic response of the intact bladder,
did not antagonize the stimulation of the toad bladder cyclase preparation by
vasopressin.
Adenosine 3',5'-cyclic monophosphate (3',5'-AMAP) appears to be anl intra-
cellular mediator in the action of neurohypophyseal hormones in amphibian skin
and urinary bladder as well as mammalian kidney.' Thus, (a) vasopressin
increases the levels of 3',5'-AMP in toad bladder;2 (b) the addition of 3',5'-AMP
mimics the effects of vasopressin to enhance water and sodium transport in the
isolated toad bladder system;3 and (c) vasopressin stimulates adenyl cyclase
activity of membrane fractions derived from kidney medullary tissue.4-6 It is
less certain that 3',5'-AMP is the exclusive intermediary in vasopressin action.
The dissociation of the neurohypophyseal hormone-induced increase of water
permeability (hydroosmotic response) and Na-flux in amphibian tissues, known
for several years,7'8 suggested the possibility that two independent hormone recep-
tor sites are involved. The finding that 3',5'-GMP stimulates Na-flux, but not
water flow,9 raises an additional possibility that vasopressin action, at least for
Na-transport, may be mediated in part by 3',5'-GMP as well as by 3',5'-AMP.
The present investigation was undertaken to measure the direct effects of
neurohypophyseal hormones and synthetic analogs upon adenyl cyclase activity
in a membrane fraction derived from toad bladder epithelial tissue. The prop-
7
Downloaded by guest on March 8, 20218 PHYSIOLOGY: BAR ET AL. PROC. N. A. S.
erties of such an adenyl cyclase preparation are described. The effects of neuro-
hypophyseal hormones and synthetic analogs upon the activity of toad bladder
cyclase have been compared and correlated with the hydroosmotic effects of
these agents in the intact toad bladder.
Preparation of Toad Bladder Adenyl Cyclase. Toads (Bufo mnarinus) used iii
these studies were purchased from Schettl Frog Farm, Minneapolis, Minn. or from
National Reagent, Inc., Bridgeport, Conn. Bladders were removed from 6-12 pithed
toads and rinsed in ice-cold amphibian Ringer's (containing 1.0 mM CaCl2, 2.0 mM KCI,
2.4 mM NaHCO3, and 111.0 mM NaCl; equilibrated with air); epithelial cells were
scraped from the mucosal surface and collected in ice-cold 0.225 M sucrose (containing
0.1 mM EDTA and 0.01 M Tris 11HC, pH 7.5). The cells were washed (a mucous layer
was removed by suction) and homogenized in sucrose medium with a "tight-fitting" glass
homogenizer and Teflon pestle. The homogenate was spun at 600 X g for 10 min and the
pellet was resuspended in 1.5 ml of the sucrose medium and centrifuged again. Distribu-
tion studies showed that the bulk of adenyl cyclase activity was associated with this 600 X g
pellet and that this fraction exhibited the greatest sensitivity to [8-arginine]-vasopressin
(AVP), [8-lysinel-vasopressin (LVP) and oxytocin (OT). This fraction, designated as
toad bladder cyclase, was used in all subsequent studies.
Assay of Adenyl Cyclase. Adenyl cyclase activities in toad bladder fractions were
assayed using methods previously described' modified as follows: assays were conducted
at pH 8.4 (the optimum for neurohypophyseal hormone action in the intact toad blad-
der)'1 using incubation periods of 20-30 min at 370C; under these conditions, ATP levels
are maintained at 70% of the initial value, with or without added hormones. EGTA (1.0
mM) was routinely added because it was observed that either EGTA or EDTA at this
concentration consistently increased hormonal stimulation without influencing basal
cyclase activity. Cold 3',5'-AMP (at 0.5 mM) was used for "trapping" the labeled
3',5'-AMP formed from ATP-a-'2P. The 3',5'-AMP formed in the toad bladder system
was authenticated by two-dimensional paper chromatography, as previously described.'2
Three toad bladder cyclase preparations were used in these studies. Each preparation
was divided into equal aliquots, rapidly frozen, and stored at -70'C. Assays were
performed after thawing, with various concentrations of test compounds using AVP
as standard. Basal and hormone stimulatable adenyl cyclase activity was retained in the
frozen state for several weeks. After thawing, the preparation exhibits increased lability
and loses about 50% of activity upon standing for 3-4 hr in an ice bath. Under basal
conditions, and in the presence of AVP and OT, 3',5'-AMP formation increased linearly
with time when observed for a 30-min period. The rate of 3',5'-AMP formation in the
presence of hormones was found to be proportional to enzyme (protein) concentration
of 100-300 jg/ml. The relative effects obtained with various neurohypophyseal pep-
tides were reproducible within a given preparation. Between different preparations there
was great variation in the degree of stimulation produced by AVP; nevertheless the pat-
tern of relative activities of the peptides was consistent.
Assay of Hydroosmotic Effects in Intact Toad Bladder Tissue. The agonistic
and antagonistic hydroosmotic properties of several neurohypophyseal peptides were
measured in the intact toad bladder by using the sac preparation of Bentley"3 as modified
by Eggena, Schwartz, and Walter.'4
Results and Discussion. In analogy with the adenyl cyclase system of the
rat fat cell membrane,'0" 5 the effects of hormones of toad bladder adenyl cyclase
may be considered in terms of a sequential set of reactions: (a) an initial step
where hormone interacts with a selectivity unit termed "receptor," (b) a sub-
sequent coupling sequence, which ultimately leads to (c) the activation of adenyl
cyclase. The receptor of toad bladder cyclase is highly specific for neurohy-
pophyseal hormones (Fig. 1). A large number of hormones known to activate
Downloaded by guest on March 8, 2021VOL. 67, 1970 NEUROPHYSEAL HORMONE AND ADENYL CYCLASE 9
z
G 60
0
50
FIG. 1.-3',5'-AMP production as L - 0
function of neurohypophyseal hormone K 40
concentration: 18-arginine]-vasopressin
(- ), [8-lysine]-vasopressin (-I-),
and oxytocin (-0--). All assays were o i
30
conducted with the same enzyme prepara- /
tion on the same day under standard 2-J 20_
conditions, with 1 mM EGTA present. ,
Protein concentration was 0.11 mg/mi. 0o-
2 01
Cf o lo lo 7 lo45 1o 3
HORMONE CONCENTRATION (MOLAR)
adenyl cyclase in other target cells or tissues, are without stimulatory or inhibi-
tory effect on the toad bladder cyclase preparation at concentrations ranging
from 1 to 100 MM; in this category we can include L-epinephrine, glucagon,
ACTH, parathyroid hormone, thyroid-stimulating hormone, and L-thyroxin.
The results in Table 1 show that the relative stimulatory activities of arginine
TABLE 1. Comparison of the hydroosmotic activity of neurohypophyseal peptides in the
intact toad bladder and their action on adenyl cyclase from bladder epithelium.
Adenyl Cyclase Assay Hydroosmotic Assay
Affinity Maximum Affinity Maximum
constant response constant response
Compound (pD2)a (% of AVP)b (pD2)a (% of AVP)b
[8-Arginine]-vasopressincd 6.9 100 9.4e 100
Oxytocinf"g 6.4 96 8.6e 100
[8-Lysinel-vasopressinc h 5.9 56 7.4 100
[8-Arginine]-vasopressinoic acidcd 6.3 80 9.1 100
De-Di-AVPf i 6.2 80 8.3 100
Oxytocinoic acidfi 5.9 60 8.5 100
[1-0-Mercaptopropionic acid, 8-
alanine]-oxypressinf"' 5.3 30 7.0k 100
[2V-0Methyltyrosinel-oxytocinf. 5.3 27 7.1*, 88
[5-Valine]-oxytocinfI10 PHYSIOLOGY: BAR ET AL. Puoc. N. A. S.
vasopressin, lysine vasopressin, and oxytocin upon the toad bladder cyclase
preparation directly correlate with the effects of these hormones in intact bladder
in terms of the "affinity" parameter, pD2 (which is a measure of hormone binding
to receptor), but not in terms of intrinsic activity-as defined by maximal 3',5'-
AMIP production by the isolated cyclase preparation or by maximal hydroosmo-
tic response in the intact toad bladder. The parallelism of the affinity parame-
ters observed with naturally occurring iieurohypophyseal peptides in both sys-
tems also holds for the structural analogs studied, which include [8-arginine]-
vasopressinoic acid, oxytocinoic acid, [5-valine1-oxytocin, an analog of [1-al-
mercaptopropionic acid, 8-arginine]-vasopressin in which both sulfur atoms have
been replaced by methylene moieties (De-Di-AVP), [2-O-methyltyrosine]-oxyto-
cin (2-O-Me-OT), and [1-ft-mercaptopropionic acid, 8-alanine1-oxypressin (De-
8-Ala-OP). It is of special interest that 2-0-Me-OT and De-8-Ala-OP (intrinsic
activity of 0.88 and 1.00, respectively, in the intact toad bladder) have about
30% of the stimulatory capacity of arginine vasopressin on the bladder cyclase
preparation. In other words, 3',5'-AMP produced by hormones and analogs at
rates in excess of that achieved with 2-0-Me-OT or De-8-Ala-OP would appear
not to be translated into a hydroosmotic response. This consideration provides
independent support for the view that the toad bladder has a "receptor reserve"
with respect to hormone-induced hydroosmotic activity. 16
Hormone analogs which inhibit the hormone-induced hydroosmotic response
of the toad bladder [1-f3-mercaptopropionic acid, 2-alanine]-oxytocin,17 [1-N-
carbamoylcysteine,2-O-methyltyrosine ]-oxytocin,l8 and [2-0-ethyltyrosine ]-oxy-
tocin"9 also inhibit arginine vasopressin-induced stimulation of the bladder
cyclase preparation; their relative antagonistic effectiveness in the intact bladder
is retained in the adenyl cyclase assay (Fig. 2).
FIG. 2.-Effect of antagonistic ineurohypophyseal
a 10O,
Do_
\ N-ts
hormone analogs on [8-arginine]-vasopressin-in-
duced adenyl cyclase activation. The percentage of
_j \ \inhibition of 1 X 10-6 M [8-argininel-vasopressin
Z 50 \ Vis plotted along the ordinate; increasing concen-
A ~~~trations of [1-ft-mercaptopropionic acid, 2-alanine]-
oxytocin (-@-), [1-N-carbamoylcysteine, 2-0-
methyltyrosine]-oxytocii (-u-), and [2-0-
0 + I- ,5 ethyltyrosine]-oxytocin (-0--) are plotted along
0 IT6 13 the abscissa. Assay procedure and protein con-
CONCENTRATION OF ANTAGONIST (MOLAR) centration were the same as described in the legend
to Fig. 1.
Despite the general parallelism of the effects of neurohypophyseal peptides in
the intact bladder and on bladder adenyl cyclase activity there are marked
quantitative differences. The peptide concentrations required to elicit half-
maximal effects on the bladder cyclase preparation are about two orders of
magnitude higher than for the intact toad bladder. The phenomenon that
higher hormone concentrations are required to elicit a half-maximal response in
isolated cyclase preparations than in intact cells has been observed in a number
of systems.10'20'21
The availability of a hormone-sensitive toad bladder cyclase preparation has
Downloaded by guest on March 8, 2021VOL. 67, 1970 NEUROPHYSEAL HORMONE AND ADENYL CYCLASE 11
made it possible to study the effects of several agents known to affect the in
vitro hydroosmotic response of the toad bladder to neurohypophyseal hormones
but not to 3',5'-AMP, for example, PGE1,22 mercaptans, and disulfides.23-25
PGE1, at concentrations ranging from 0.1 to 100 ,ug/ml, was found to be without
significant inhibitory effect on either basal or arginine vasopressin-stimulated
cyclase activity. The inhibitory effect of PGE1 in the intact toad bladder, which
appears to be noncompetitive,16 may involve selective sites for PGE1 which in-
fluence, but are not part of, the isolated toad bladder cyclase system. Similarly,
the stimulatory activity of De-Di-AVP (a highly active neurohypophyseal hor-
mone analog which, unlike the disulfide-containing hormones, cannot participate
in a thiol-disulfide interchange reaction) was not affected by 1 mM oxidized or
reduced glutathione or by dithioerythritol; nor did these agents significantly
influence basal cyclase activity. These results suggest that the mercaptan or
disulfide inhibition observed in the intact bladder system is not achieved by
direct interaction with the hormonal receptor or the catalytic unit of the cyclase
system.
The present studies have involved a particulate adenyl cyclase preparation
derived from the multiple cell types which comprise the toad bladder epithelium.
Independent of uncertainties concerning whether there is more than one type of,
or more than one locus for, neurohypophyseal hormone receptors in the intact
toad bladder, there is a striking correlation of peptide effects on the adenyl
cyclase preparation derived from the multiple cell types of bladder epithelium
and the hydroosmotic response in intact tissue.
The authors are grateful to Dr. J. Nirmul and Miss A{. Wahrenburg for performing studies
with the intact toad bladder.
Neurohypophyseal hormones are denoted in accordance with the IUPAC-IUB Tentative
Rules (Biochemistry, 6, 362 (1967)).
*
This study was supported in part by U.S. Public Health Service grants AM-10080 and
AM-13567 of the National Institute of Arthritis and Metabolic Diseases and by the U.S.
Atomic Energy Commission. It was presented in part at the colloquium on Role of Adenyl
Cyclase and Cyclic 3',5'-AMP in Biological Systems at the Fogarty International Center of the
National Institutes of Health, Bethesda, Md., November 17-19, 1969.
t Present address: Department of Pharmacology, University of Alberta, Edmonton,
Alberta, Canada.
t Requests for reprints may be addressed to Dr. 0. Hechter. Present address: Depart-
ment of Physiology, Northwestern Medical School, 303 East Chicago, Chicago, Ill. 60611.
1 Robison, G. A., R. W. Butcher, and E. W. Sutherland, Ann. Rev. Biochem., 37, 149 (1968).
2 Handler, J. S., R. W. Butcher, E. W. Sutherland, and J. Orloff, J. Biol. Chem., 240, 4524
(1965).
3 Orloff, J., and J. S. Handler, J. Clin. Invest., 41, 702 (1962).
4Brown, E., D. L. Clarke, V. Roux, and G. H. Sherman, J. Biol. Chem., 238, PC852 (1963).
5 Chase, L. R., and G. D. Aurbach, Science, 159, 545 (1968).
6 Dousa, T., 0. Hechter, R. Walter, and I. L. Schwartz, Science, 167, 1134 (1970).
7Bentley, P. J., J. Endocrin., 39, 299 (1967).
8 Bourguet, J., and J. Maetz, Biochim. Biophys. Acta, 52, 552 (1961).
9 Bourgoignie, J., S. Guggenheim, D. M. Kipnis, and S. Klahr, Science, 165, 1362 (1969).
10 Bar, H.-P., and 0. Hechter, Proc. Nat. Acad. Sci. USA, 63, 350 (1969).
' Gulyassy, P. F., and I. S. Edelman, Biochim. Biophys. Acta, 102, 185 (1965).
12 Bar, H.-P., and 0. Hechter, Anal. Biochem., 29, 476 (1969).
13 Bentley, P. J., J. Endocrin., 17, 201 (1958).
14 Eggena, P., R. Walter, and I. L. Schwartz, J. Gen. Physiol., 52, 465 (1968).
Downloaded by guest on March 8, 202112 PHYSIOLOGY. BAR ET AL. PROC. N. A. S.
16 Birnbaumer, L., and M. Rodbell, J. Biol. Chem., 244, 3477 (1969).
6 Eggena, P., I. L. Schwartz, and R. Walter, J. Gai. Phy8iol., 56, 250 (1970).
17 Walter, R., and I. L. Schwartz, Life Scieswe, 7, 545 (1968).
18 Chimiak, A., K. Eisler, K. Jobt, and J. Rudinger, Collect. Czech. Chem. Commun., 33, 2918
(1968).
19 Zhuze, A. L., K. Joit, E. Kasafirek, and J. Rudinger, Collect. Czech. Chem. Commun., 29,
2648 (1964).
'0 Gilman, A. G., and T. W. Rall, J. Biol. Chem., 243, 5867 (1968).
21 Pastan, I., and R. Katzen, Biochem. Biophy8. Aeg. Commun., 29, 792 (1967).
22 Orloff, J., J. S. Handler, and S. Bergstr6on, Nature, 205, 397 (1965).
2" Bentley, P. J., J. Endoorin., 30, 103 (1964).
24Handler, J. S., and J. Orloff, Amer. J. Physiol., 206, 505 (1964).
2" Rasmussen, H., I. L. Schwartz, R. Young, and J. Marc-Auriele, J. Gen. Phyeiol., 46, 1171
(1963).
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