CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
FLUCTUATION OF CYCLIC AMP - BINDING SITE
OCCUPANCY AS A CONSEQUENCE OF HORMONE - DEPENDENT
CYCLIC
&~P
FOR}lATION IN RAT RENAL CORTEX
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Gregory Alan Arvesen
January 1982
The Thesis of Gregory Alan Arvesen is approved:
Richard L. Potter, Ph.D.
Chairman
California State University, Northridge
TABLE OF CONTENTS
Page
LIST OF TABLES... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
~v
LIST OF FIGURES..............................................
~v
ABSTRACT.....................................................
v
Chapter
1
INTRODUCTION ....................................... .
1
2
MATERIALS AND NETHODS .............................. .
4
3
RESULTS ............•................................
11
Cyclic ANP-binding characteristics of
renal cortex cytosol.............................
Effect of incubation with hormone on
unoccupied cANP- binding sites..................
Time course of unoccupied binding site
depletion........................................
Effect of phosphodiesterase inhibitors
on unoccupied binding sites......................
11
15
19
19
DISCUSSION ......................................... .
28
LIST OF REFERENCES...........................................
36
4
LIST OF TABLES
Table
1
2
3
Page
Effect of PTH on cyclic AMP and cAMP Receptor
Content in Renal Cortex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
Effect of PTH on cyclic AMP Binding Affinity
in Renal Cortex Slices · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
18
Reduction of Unoccupied cyclic AMP Binding
Sites in Renal Cortex by Various Agents •................ ·
27
LIST OF FIGURES
Figure
Page
1
Binding of [3H]-cAMP to renal cortex cytosol .•...........
13
2
Effect of parathyroid hormone on cyclic fu~
(cAMP) formation and unoccupied cyclic AMP
binding site concentration ...............................
17
Effect of prostagland in E1 on cyclic AMP (cAMP)
formation and unoccupied cyclic ANP binding site
concentration............................................
22
Effect of parathyroid hormone, epinephrine and
prostaglandin E1 on cyclic AMP (cAMP) formation
and unoccupied binding site number .......................
24
Time course of parathyroid hormone-induced
cyclic A}IP (cAMP) formation and unoccupied
binding site depletion and restoration ...................
26
3
4
·s
iv
ABSTRACT
FLUCTUATION OF CYCLIC AMP BINDING SITE
OCCUPANCY AS A CONSEQUENCE OF HORMONE-DEPENDENT
CYCLIC AMP FORMATION IN RAT RENAL CORTEX
by
Gregory Alan Arvesen
Master of Science in Biology
The initial event in cyclic AMP action
~s
thought to be binding to
the regulatory subunit and subsequent activation of protein kinases. As
a measure of hormone action distal to cyclic AMP generation, cyclic AMP
binding to postmicrosomal supernatant fraction from rat renal cortex
was studied.
Renal cortical slices were incubated under a variety of
conditions in Krebs-Ringer bicarbonate buffer and cyclic AMP binding by
100,000 x g supernatant fraction was determined by trapping [3H]-cAMP protein complexes on nitrose cellulose filters. Binding characteristics
were
evaluated
by
the method
of
Scatchard.
After correcting
for
nonspecific binding, results were consistent with binding by a single
class of sites with an apparent binding constant of 39 nH.
There were
1.56 + .20 x 1012 sites per milligram protein and treatment with PTH or
PGE1 in doses insufficient to cause significant elevation of cyclic AMP
content, decreased these unoccupied binding sites.
Affinity for cyclic
AMP was the same regardless of the degree of binding site depletion or
mode
of
cyclic
AMP
elevation.
Maximal
unoccupied
binding
site
depletion was not increased by additional agonist, despite further
v
increases
in
cyclic
AMP
formation.
Fluctuations
~n
physiologic
concentrations of hormone may therefore regulate cellular activity by
inducing only modest changes in intracellular cyclic AMP.
Time course
studies showed that unoccupied binding site number follows changes
cyclic
&~P
~n
concentration with a nadir at 10 minutes and restoration to
basal levels by 45 minutes.
At maximally effective concentrations of
PTH and PGel cyclic AMP formation was additive, but unoccupied binding
site depletion was not greater than that seen with either agonist alone.
Persistence of 35% of unoccupied binding sites despite maximal stimulation of cyclic AMP by hormone suggests a physiological role in the
binding equilibrium.
Depletion of cyclic
&~P
binding sites appears to
fluctuate as a direct consequence of elevations in ambient cyclic AMP
concentration and does not appear to be independently regulated.
vi
Chapter 1
INTRODUCTION
Since
the
discovery by
Sutherland
glucogenolytic effect of epinephrine on
formation of adenosine 3 1
,
51
-
and
Rall
liver
H
(1960)
that
the
mediated by
the
cyclic monophosphate (cyclic AMP),
there have been numerous reports identifying several hormonal systems
in which the hormonal responses in the target cells are mediated by a
prior increase in cyclic AMP.
mimic
Because cyclic AMP has been shown to
the physiologic effects of the hormone
tissues and because peptide hormones are
penetrate
the cell,
~n
thought
hormone-sensitive
to be unable
to
the concept of cyclic AMP acting as a second
messenger has become widely accepted (Haynes et al., 1960; Chase and
Aurbach, 1968; Dousa and Rychlik, 1968; Robison et al., 1968, 1971;
Major and Kilpatrick, 1972; Beall and Sayers, 1972).
Many studies have implicated cyclic AMP as a primary mediator of
parathyroid hormone (PTH) and prostaglandin E1 (PGEl) actions on renal
cortex (Chase and Aurbach, 1968; Aurbach and Chase, 1970; Melson et al,
1970; Aurbach et al., 1972; Beck et al., 1972a; Kurokawa and Hassry,
1973; Kurokawa et al., 1974; Birnbaumer and Yang, 1974; Chase, 1975;
Lorentz, 1976; Morrison et al, 1976).
Effects of hormonal action on nephron functions located in the
proximal tubule, such as gluconeogenesis and reabsorption of sodium,
bicarbonate, phosphate, calcium, water and amino acids (Krebs et al.,
1
2
1963; Pagliara and Goodman, 1969; Kaminsky et al., 1970; Nagata and
Rasmussen, 1970 a, b; Rasmussen and Nagata, 1970; Agus et al., 1971;
Aurbach et a1., 1972; Agus et al., 1973; Kurokawa et al., 1973; Kurokawa
and Massry, 1973; Roobol and Alleyne, 1973; Crumb et al., 1974; Short et
al., 1974; Diaz-Buxo et al., 1975; Borle and Uchikawa, 1978) have been
shown to be mediated through the stimulation of membrane-bound adenyl
cyclase (Chase and Aurbach, 1968; Aurbach et al., 1972; Becket al.,
1972b; Sutcliffe et al., 1973) which subsequently increases cyclic AMP
concentration within the renal parenchyma (Nagata and Rasmussen, 1968;
Aurbach et al., 1969).
It is generaly acknowledged that the exclusive role of the cyclic
nucleotide in eukaryotic cells is to activate the catalytic subunit of
protein kinase (Kuo and Greengard, 1969; Gill and Garren, 1971; Robison
and Sutherland, 1971).
Cyclic AMP forms a complex with the regulatory
subunit of protein kinase, causing its dissociation from the holoenzyme
and thereby activating the catalytic subunit (Gill and Garren, 1970;
Kumon et al., 1970; Tao et al., 1970; Brostrom et a1., 1971; Chambaut et
al. , 1971; Erl ichman et al. , 1971; Garren et al. , 1971; Reimann et al.,
1971; Kumon et al., 1972).
Thus, cyclic AMP generated as a result of
hormone stimulation causes an activation of in vivo phosphorylation.
Evidence from a variety of tissues indicates that full stimulation
of protein kinase activity may be achieved by cyclic AMP concentrations
considerably below maximally stimulable levels (Beall and Sayers, 1972;
Williams, 1972; Catt and Dufau, 1973; Dufau et a1., 1973; Mendelson et
3
al.,
1975; Moyle et al., 1975; Sharma et al., 1976). Consequently,
changes in occupancy of regulatory subunit may reflect the physiologic
response of hormone action more accurately than do alterations 1.n
cyclic nucleotide concentration.
In order to define further the role of
hormone-sensitive adenylate cyclase systems in renal cortex function,
this
study investigated the effect of hormone
stimulation on
the
occupancy of cyclic AMP binding sites on the protein kinase regulatory
subunit.
The
present
study characterizes
the
cyclic
AMP-binding
·properties of renal cortex cytosol, and describes the changes in such
binding that are induced by hormonal stimulation.
Chapter 2
MATERIALS AND METHODS
Partially purified bovine parathyroid hormone (TCA precipitate,
500 U.S.P. Units/mg) was the gift of Dr. Henry Keutmann.
was dissolved
The hormone
in 0.005 N acetic acid and stored frozen in small
aliquots. Prostaglandin E1 was provided by the Upjohn Corp., and was
dissolved in ethanol.
[3H]-c&~P (37.7 Ci/mmol) was purchased from New
England Nuclear Corp.
Other reagents and chemicals were of analytical
grade and were purchased from standard suppliers.
Incubation of Kidney Slices 1n Vitro
Female
Sprague
cervical dislocation,
Dawley rats,
200-250
grams were killed by
and kidneys were placed into chilled 0.25 M
Slices of renal cortex, approximately 0.5 mm thick, were
sucrose.
weighed
-
and
placed
1n
iced
sucrose
for
30 minutes.
After
the
equilibration period the slices were transferred to flasks on 1ce
containing 10 ml Krebs-Ringer-Bicarbonate buffer (KRB) equilibrated
with 95% 02-5% C02, and containing 2 mg/ml glucose and 1.0 mg/ml bovine
serum albumin.
Incubations were initiated by adding the appropriate
concentration of hormone or drug while on ice and transferring the
flasks to a 37°C Dubnoff metabolic shaker.
Incubations were terminated
by removing the slices to chilled buffer and homogenizing (Polytron,
Brinkman Industries).
cyclic
ANP
Extraction and preparation of the tissue for
determination or measurement of unoccupied
4
cyclic
&~P
5
receptor concentration was carried out exactly as described below.
Cyclic AMP Determination
Incubations were terminated by transferring blotted slices to
chilled 6% TCA ( 2 ml),
and homogenizing
(Polytron,
Brinkman Ind-
ustries). 4000 dpm [3H]-cAMP were added for recovery, and the homogenate was centrifuged at 3000 x g for 20 minutes.
The supernatant
fraction was extracted three times into equal volumes of H20-saturated
ether. Residual ether was removed from the aqueous phase under a stream
of N2, and the extract was neutralized with 25 ul concentrated NaOH. To
the neutralized extract was added 0.1 ml each of 5% ZnS04 and 0.3 N
Ba(OH)2; the mixture was centrifuged, and the supernatant fraction was
applied to a 2.5 ml column of Dowex 50 resin (HCL form) as described in
Marcus
and Orner (1977).
The cyclic AMP containing column fractions
were collected and lyophillized,
and
the residue
dissolved
acetate buffer, pH 4.0 for determination of cyclic AMP.
1.n Na
Recovery of
[3H]-cAMP approximated 40%.
Cyclic AMP was measured by a radioligand assay as modified from
Tsang et al., (1972).
A crude cytosol fraction of bovine thyroid gland
was used as the cyclic AMP binding protein (Orgiazzi et al., 1975).
Each tube contained 0.01 M sodium acetate buffer (pH4.0), 0.45mM EDTA,
lmM theophylline, approximately 40,000 dpm [3H]-cAMP, binding protein
sufficient to bind 50% of added radioactivity, and 200 ul unknown sample
or cold cyclic AMP standard in a final reaction volume of 1 ml.
After
6
incubation at 4°C for
90 minutes,
bound
and
free
[ 3H] -cAMP were
separated by addition of 1 ml Dowex AG1X2 resin (chloride
suspended in an equal volume of chilled· acetate buffer.
form),
After an
additional 20 minutes at 4°C the tubes were centrifuged, and aliquots of
the supernatant fractions were taken for liquid scintillation spectroscopy, using ACS (Amersham) as the scintillation cocktail.
Significant displacement of the radioligand was achieved with 1
pmole cyclic AMP, and the standard curve afforded satisfactory measurement of cyclic ANP over the range of 1-40 pmoles.
Coefficient of
variation of the assay was 13% for 1-10 pmoles, and 7.8% for the 20-80
pmoles.
Relative to cyclic ANP, the ratios of equilibrium constants of
association for the binding protein were 7.3 x 10-3M and 6.1o-6M for
cyclic GMP and ATP, respectively (Korenman, 1970).
96% of cyclic AMP
measured was destroyed by incubation with beef heart phosphodiesterase.
Determinations of cyclic AMP for each sample were carried out in
duplicate, and the results are expressed as picomoles cyclic AMP per
milligram of protein, and represent the mean of four or more samples, as
indicated.
Results
are
expressed
significance was estimated using
the
as
mean
+
two-tailed
SEM.
Statistical
Student's
t-test.
Protein was determined by the method of Lowry et al., (1951).
Measurement of Cytosol Unoccupied cAMP-receptor Concentration
Incubations were stopped by transferring the slices to two volumes
7
of neutral ethylenediaminetetraacetic acid (EDTA), lmM, and homogenizing (Polytron, Brinkman Industries; three bursts of 30 sec.).
Homogenates were centrifuged at 20,000 x g for 15 minutes, and
supernatant fractions were centrifuged for one hour at 100,000 x g in a
Beckman ultracentrifuge (model L-2) to yield the cytosol fraction.
Unoccupied binding sites are defined as those which are not occupied by
endogenous cyclic AMP, and are therefore available to bind [3H]-cAMP ~n
the radioligand binding assay similar to that reported by Dufau et al.,
(1977)
as modified
by Marcus
et
al.,
(1979).
Duplicate reaction
mixtures of 0.2 ml were prepared by rapidly adding 30 ug of cytosol
protein to 0.05 M sodium acetate (pH5.0); 10mM MgCl2;
5nu~
theophylline;
9.3 nM [3H]-cAMP (approximately 40,000 cpm); and concentrations of
unlabeled cAMP varying from 5nM to 2.5uM.
The mixtures were incubated
for 20 minutes at room temperature, and protein-bound [3H]-cAMP was
separated from the free by filtration onto membrane filters.
Filters (Millipore, 0.45 microns) were pre-soaked for 30 minutes
in 0.025M Tris-HC1, pH 7.4, containing 10 mM EDTA and 10 mM MgCl2. Two
ml ice-cold .05 M acetate buffer, (pHS.O), was added to each tube, and
the contents were applied to a reservoir above the filters. After one
minutes vacuum was applied, and the samples were drawn through and
washed three times with 2 ml of the same cold buffer. The filters were
dried under a heat lamp,
and were then immersed
~n
toluene-based
scintillation fluid (ACS, Amersham).
Radioactivity was determined by
liquid scintillation spectroscopy.
Radioactivity remaining on the
8
filters represented [3H]-cAMP bound to protein.
described,
displaced
mixtures.
over
by
85%
adding
of
radioactivity
excess
Background
unlabeled
binding,
was
specifically
cyclic
obtained
Under the conditions
by
AMP
to
bound
the
processing
and
reaction
incubation
aliquots in the absence of protein, was negligible.
Total cyclic AMP binding capacity refers to unoccupied sites plus
those occupied by endogenous cyclic AMP, determined according to the
radioactive exchange method of Harbon et al
(1976).
After homo-
genization and centrifugation at 100,000 x g, cytosols were maintained
at 4° C for 60 minutes in the presence of 2 x 10-7M unlabeled cyclic AMP,
an excess of nucleotide sufficient to saturate all specific binding
sites.
The cytosols were then incubated at room temperature with 2uM
cyclic AMP containing approximately 200,000 cpm [3H]-cAMP, 0.1 mM ATP
and 10 mM MgC1z.
Under these conditions, exchange has been shown to be
virtually complete by 30 minutes Harbon et al., (1976).
Incubation
mixtures were then passed over Millipore filters as outlined, and bound
[3H]-cAMP was measured.
Preliminary experiments indicated that binding by cytosol receptor
which had been saturated with [3H]-cAMP was stable for 60 minutes at
room temperature,
after which it decreased.
Under the conditions
outlined, exchange between [3H]-cAMP and bound cyclic AMP was complete
by 10 minutes, and stable for 60 minutes.
Therefore, exchange reactions
were carried out for 10, 30, and 60 minutes, and the mean+ SEM of bound
radioactivity at equilibrium was used to compute the total binding site
9
number.
Data presentation
Unoccupied cyclic AMP binding sites were computed by plotting the
ratio of bound/free as a function of bound according to the method of
Scatchard
(1949).
Each plot was
subtracting non-specific binding.
composed of
five
points,
after
Non-specific binding was defined as
radioactivity bound in the presence of 2.5 uM unlabeled cyclic AMP.
Binding characteristics were determined by linear regression analysis
of the Scratchard plots, where the x-intercept equals the number of
available binding sites and the slope represents binding affinity.
concentration of
available binding sites was
calculated
The
from the
following equation:
Binding sites/mg protein
=
Mx X A/m
Where Mx = moles bound at x-intercept
A
Avogadro's number
m
milligrams protein per reaction mixture
Statistical comparison of slopes and intercepts of linear regression
data were
computed
by
standard
statistical
methods
Snedecor
and
Cochran, (1972).
Analysis of binding by linear regression of Scatchard plots has
been applied widely to receptor-ligand studies Rodbard,
(1973). The
10
validity of this approach may be limited by several factors, including
cooperative interaction between receptors and non-uniformity of variance at different points along the Scatchard plot (Rodbard, 1973).
In
the present studies no evidence of cooperativity emerged: 1) Scatchard
diagrams were linear:
2) When binding sites were depleted by hormone
incubation, analysis of residual binding gave slopes identical to those
found in the basal state (Table 2).
Moreover, calculation of variance
for values of B/F across the entire range of the plot demonstrated
reasonable uniformity (range of Sy
=
3.75 + .09 x 10 -7), and there was
no correlation of variance with the independent variable, x.
There-
fore, linear regression provides a reasonable approximation of the two
parameters of interest,
slope and x-intercept,
and
the confidence
limits for each can be calculated by standard methods (Rodbard, 1973).
Chapter 3
RESULTS
Cyclic AMP-binding characteristics of renal cortex cytosol
Binding of [ 3H] -cAMP to cytosol prepared from renal cortex is
illustrated
~n
Figure 1.
Scatchard analysis of data resolves two
components, one of a single class of high affinity- low capacity (Kd
3.87 x 10-8; r
=
=
0.992), the other of low affinity and unsaturable.
Nonspecific binding was 3.6% of the maximal bound to free ratio (B/F)
when
the
nonspecific component was
extrapolated
to
the ordinate.
Subtraction of nonspecific binding allows extrapolation of the Scatchard diagram to the abscissa by linear regression and shows maximal
specific
binding
of
1.24
picomoles
per
reaction mixture.
This
corresponds to 1. 39 x 1012 unoccupied binding sites/mg protein.
eight separate experiments, the mean
(~
In
SD) for the basal number of
unoccupied binding sites was 1.56 ~ 0.20 x 1012/mg protein (range, 1.31
- 1.85). The mean
z
Ci
SD) of basal Kd for these same experiments was 4.19
0.45 x 10-8M (range 3.64- 4.75 x lo-8 M).
The total cyclic AMP - binding site concentration of renal cortex
cytosol in the basal state was 3.25
z
0.34 x lol2fmg protein (Table 1).
Unoccupied binding site content in the same experiment was 1.74
x lol2/mg protein, which is 53.3% of the total.
11
~
0.01
12
Figure 1. Binding of [3H]-cAMP to renal cortex cytosol.
The amount of
nucleotide bound (B) 1s plotted by the Scatchard method as a
function of the ratio of the concentration of bound to free
cyclic AMP (B/F).
The concentration of free cyclic AMP
(cAMP) was obtained by subtracting the cyclic AMP bound from
the total initial cyclic AMP concentration.
Renal cortex was
incubated in Krebs-Ringer Bicarbonate buffer for 2.5 minutes
and cytosol was prepared as described in Methods.
Binding
was assayed by rapidly adding 600 ng of cytosol protein to the
cyclic nucleotide binding mixture (described in Methods) and
concentrations of unlabeled cyclic AMP between 5 nM and 2.5
nM.
Mixtures were incubated for 20 minutes at room temp-
erature and isolation of protein-bound [ 3H] -cAMP was performed as described in the text.
Scatchard analysis of data
resolves two components, one of high affinity-low capacity,
the other of low affinity and nonsaturable.
represents a single determination.
Each point
[3H]-c&~P bound by the
high affinity component was displaced by unlabeled
represents specific binding.
nonspecific.
ch~P
and
The nonsaturable component 1s
Solid line represents a least squares fit of
original data; broken line represents specific binding (total
minus nonspecific)
extrapolated to x-intercept by linear
regression.
Binding of cyclic AMP to cytosol protein
Inset:
as a function of the total cyclic AMP concentration added.
Each point represents the mean of three determinations.
13
0.1
'il:
::::!E
~~
:::Jtn
0;!!
CDO
IJ.J
I..U
E
0.!2
~
a::
.....
0
z
0.4
1.1..
=>
m
0
0.08
0.04
0.4
0.8
12
1.6
pmoles cAMP BOUND
2.0
2.4
TABLE 1.
EFFECT OF PTH ON CYCLIC AMP AND cA.1vlP RECEPTOR CONTENT IN RENAL CORTEX
Incubation
Conditions
Control
PTH, 16 U/ml
cyclic AMP
Binding Sites
(1o12Jmg protein)
Total
Unoccupied
(pmoles/mg protein)
5.48 :!:. 0.25
16.03 !. 1.84
3.25 .:!:. 0.34
3.12 ~ 0.33
l. 74 ~ 0.01
1.11 ~ 0.01
% Unoccupied
53.5
35.6
Kidney slices were incubated in Krebs-Ringer Bicarbonate for 2. 5 minutes in the absence or
presence of the indicated concentration of parathyroid hormone (PTH) and prepared and assayed for
cyclic AMP and cyclic AMP binding sites according to the methods described.
the mean~ SEM of four kidney slice determinations.
computed as described in the text.
Results for cyclic AMP are
Unoccupied binding site number (mean! SEM) were
Total binding site results are the mean~ SEM of ten determinations.
,......
+:-
15
Effect of incubation with hormone on unoccupied cAMP - binding sites
Incubation of renal cortical slices with parathyroid hormone (PTH)
resulted in an increase in the cyclic AMP content and a reduction in
unoccupied binding sites (Figure 2).
A detectable elevation in cyclic
AMP content (P(0.01) was caused by 0.05 u/ml PTH and half maximal
stimulation was achieved at 1 u/ml.
Maximum reduction of unoccupied
binding sites was obtained by the lowest PTH dosage used, 0.05 u/ml.
Maximal PTH-dependent depletion represented a 38% reduction from basal
levels.
cyclic
Results
Further increases in PTH concentration resulted in greater
&~P
accumulations, but no further depletion of binding sites.
from Scatchard analysis
show that reduction in unoccupied
binding site number was not accompanied by a change in the affinity of
the residual binding sites for the nucleotide (Table 2).
Slices
of
renal
cortex incubated with PTH
~n
concentrations
greater than required for maximal cyclic AMP formation caused a 36%
reduction of unoccupied binding sites from basal levels; however, no
significant alteration in total cyclic AMP binding site concentration
was observed (Table 1). This reduction corresponds to occupancy of only
20% of the total cyclic AMP binding sites available even in the face of
greatly elevated cyclic AMP concentration.
Incubation of renal cortex with prostaglandin E1
promoted cyclic
(Figure 3).
k~P
(PGE1) also
formation and reduced binding site availability
Significant elevation in cyclic AMP production occurs at
16
Figure 2. Effect of parathyroid hormone on cyclic
and unoccupied cyclic
&~P
&~P
(cAMP) formation
binding site concentration. Kidney
slices were incubated in Krebs-Ringer Bicarbonate containing
the indicated concentrations of parathyroid hormone (PTH) for
2. 5 minutes and prepared and assayed as described in the
methods.
Binding
described Ln text.
site
numbers
(o)
Results for cyclic
were
&~P
calculated
as
levels (e) are the
mean+ SEM of four kidney slice determinations.
•
cAMP
0
BINDING SITES
2
20
'0
3'
0
(I)
V>
()
];>
~
'1)
0
......
3
0
10
0
<C
'1)
:0
0
-1
rn
z
0
0.1
1.0
PTH (UNITS/ ml)
10.0
18
TABLE 2.
EFFECT OF PTH ON CYCLIC AMP BINDING AFFINITY IN RENAL CORTEX SLICES
Incubation
Conditions
Control
PTH, 0.1 u/ml
PTH, 0.5 u/ml
PTH, 1
u/ml
Unoccupied cyclic AMP
Binding Sites
(x1o12jmg protein)
1.36 :t.
0.82 ±
0.85!.
0.86 ~
0.07
0.01
0.01
0.01
*
*-.':
K
d
Cx lo-8~.'1)
4.01
4.07
4.33
4.34
Correlation
Coefficient ( r)
0.996
0.968
0.986
0.998
Kidney slices were incubated in Krebs-Ringer Bicarbonate for 2.5
minutes in the absence or presence of the indicated concentration of
parathyroid hormone (PTH).
Unoccupied binding site number (mean :_
SEM) and binding affinity characteristics \vere computed as described
~n
the text.
Asterisk (*) indicates different from control determined
by analysis of variance (P< 0.001).
19
luM where stimulation is half-maximal.
sites was detected at 10 nM,
Maximal depletion of binding
the lowest concentration tested,
represented a 37% reduction from basal levels.
and
No changes in binding
affinity of kidney slice cytosol incubated with PGE1 were observed.
Under conditions in which cyclic AMP responses to PTH, PGE1, or
epinephrine (EPI) were maximal, the condition of PTH and PGE1 in the
absence or presence of EPI resulted in increased cyclic AMP formation
but additional depletion of unoccupied binding sites did not occur
(Figure 4).
Time course of unoccupied binding site depletion
Incubation of kidney slices with PTH resulted in a time dependent
increase
~n
2.5 minutes.
cyclic AMP concentration which was at maximal elevation at
The return of cyclic AMP content to basal levels occured
after 45 minutes (Figure 5).
Increased binding site occupancy was
detected at 1 minute with maximal depletion at 10 minutes.
Restoration
of basal level binding sites was complete by 45 minutes and was not
impaired by carrying out the entire incubation in the presence of the
protein synthesis inhibitor cyclohexamide.
Effect of phosphodiesterase inhibitors on unoccupied binding sites
Cortical kidney slices were incubated in the absence or presence
of phosphodiesterase inhibitors or hormones (Table 3).
PTH caused
significant elevation of cyclic AMP accumulation in kidney slices (P<
20
.001) while other treatments only caused slight increases between 2060% of basal levels.
All treatments produced significant increases in
cyclic AMP production and depletion of unoccupied binding sites without
altering affinity of binding indicating that cyclic AMP content and not
its manner of formation is responsible for decreased binding site
availability.
21
Figure 3. Effect of prostagland in E1 on cyclic AHP (cAHP) formation and
unoccupied cyclic AHP binding site concentration.
Kidney
slices were incubated in Krebs-Ringer Bicarbonate containing
the indicated concentrations of prostaglandin E1 (PGEl) for
2. 5 minutes and prepared and assayed as described
methods.
described
t
Binding
~n
text.
site
numbers
(o)
were
~n
calculated
the
as
Results for cAHP levels (e) are the mean
SEH of four kidney slice determinations.
22
0
cAMP
0
BINDING SITES
20
~
(/)
3
w 1.5
1U5z
0
0
0
<.!)Uj
en';
3:
"'0
........
3
10
t-
a-
wX
0..
0
1.0
0
::::>
l>
1.0
01
<..)
0
0
a..E
::::E<1!::!
--
(/)
(')
Zl-
iSO
zo:
(p
""0
:::0
0
-1
I"TT
z
05
u
0
z
::::>
0
8
7
6
-log (PGE 1 ) M
5
4
3
23
Figure 4. Effect of parathyroid hormone, epinephrine and prostaglandin
E1 on cyclic AMP
number.
(cAl~P)
formation and unoccupied binding site
Kidney slices were incubated in Krebs-Ringer Bicar-
bonate buffer for 2.5 minutes in the absence or presence of 10
u/ml parathyroid hormone (PTH), 0. 1 ml'-'l epinephrine (EPI) and
0.1
ml'-'1
prostaglandin e1 (PGEl) in the combinated indicated.
Unoccupied binding site number and cyclic AMP concentration
were determined as described in the methods.
eye lie AI1P represent mean
determinations.
.:.!;.
SEM
for
four
Results for
kidney
s 1 ice
24
pmoles cAMP/ mg PROTEJN
...1
ID
~
z
1-
8
d
(NI3..LO?.:Id 5w ; 21 _0IX )
S3..LIS 8N!GNI8 dVfi/~ G3!dfl~~ONn
25
Figure 5. Time course of parathyroid hormone-induced cyclic
fu~P
(cAJ1P)
formation and unoccupied binding site depletion and restoration.
Kidney slices were
incubated in Krebs-Ringer
Bicarbonate containing 1 mH cyclohexamide and 16 u/ml parathyroid hormone for the times indicated.
Unoccupied binding
site (o) and cyclic AJ!P concentration (•) were determined as
described
represent
in
the
mean~
text.
Results
for
cyclic Al1P
SEM for four slice determinations.
levels
26
•
0
cAMP
BINJING SITES
25
1.5
"'
"'
zz
/0
lW
/
1-
<.!)-
~!
ow
zl-o
aJa::
a.. a..
~0'1
. //
p/
/
"'C
/
3
g_
(tl
0
\ \r _..,. /
~
1.0
<5~
IJ)
(')
)>
3:
iJ
-
3
<Q
0~
Wt
-o
a..::JX
u-
!5
iJ
:::0
0
-1
r!!
z
(.)
0
z
::>
0.
lO
5
10
0
20
30
40
MINUTES
-
-~-------
~-
---~-~~---
-------~--
50
27
TABLE 3.
REDUCTION OF UNOCCUPIED CYCLIC AHP BINDING
SITES IN RENAL CORTEX BY VARIOUS AGENTS
Incubation
cyclic ANP
Conditions
(pmoles/mg protein)
.84 .± 0.57
13.41 ± 1. 50
7.64 ;!:. 0.90
5.73 + 1.43
7.63 ± 1. 35
7.65 + 1.06
Control
PTH, 10 U/ml
PGE1, 10-4}1
EPI, 10-4M
HIX, 1 rru'v!
THEO, 10 mM
~~
...,·~
..k
..k
"i':
Unoccupied cAHP
Binding Sites
(x1ol2/mg protein)
K
d
Cxlo-8H)
1. 67 ± 0.05
1. 08 ± 0.01 *
1. 24 + 0.01 -;'(
1. 36 + 0.01 -;':
1. 22 ::!: 0.01 ./(
1.14 + 0.01 t'(
4. 75
4.59
4.15
4.87
4.51
4.18
Kidney slices were incubated in Krebs-Ringer Bicarbonate for 2.5
minutes in the absence or presence of the indicated concentration of
parathyroid hormone (PTH), prostaglandin E1 (PGE1), epinephrine (EPI),
methylisobutylxanthine (MIX) or theophylline (THEO) and prepared and
assayed for cyclic M1P and cyclic AMP binding sites according to the
methods described.
Results for cyclic AHP are the mean ! SEM of four
kidney slice determinations.
Unoccupied binding site number (mean!
SEN) and binding affinity were computed as described in the text.
Asterisk
(*)
indicates
different
analysis of variance (P ( . 005) .
from control
as
determined
by
Chapter 4
DISCUSSION
It is generally acknowledged that the effects of cyclic AMP in
eukaryotic cells appear to be related to the ability of the nucleotide
to activate cyclic AMP - dependent protein kinase,
the activation
occuring as a consequence of the nucleotide binding to the regulatory
subunit of the holoenzyme (Gill and Garren, 1971; Erlichman et al.,
1971; Reimann et al., 1971).
The results presented here describe the
characteristics of the binding of cyclic
k~
to postmicrosomal super-
natant fraction prepared from rat renal cortex.
Several
tissues
possess multiple cyclic AMP binding proteins
(Knight, 1975; Talmadge et al., 1975; Sanborn et al., 1973), detected as
nonlinear Scatchard (1949) plots, indicative of binding to more than a
single class of binding sites or binding by interacting sites (Sanborn
et al., 1973).
These were not observed in renal cortex cytosol in which
Scatchard plots indicated a single order of binding sites.
In studies
with brown fat, Knight (1975) found two kinetically distinct classes of
cyclic AMP binding,
and demonstrated that incubation with catecho-
lamines altered binding affinity.
Marcus et al. (1979) reported no
changed in cyclic AMP binding affinity upon incubation of bone cells
with PTH or PGE1.
Kidney cortex
appears more closely to resemble the
bone cell, s1nce linear Scatchard plots maintained and exposure to
either PTH or PGE1 did not alter the apparent binding affinity for
cyclic AMP (Table 2).
Thus, while unable to conclude cyclic AMP binding
28
29
by renal cortext resides with a single protein species, if multiple
forms exist in kidney their affinities for the nucleotide are markedly
similar.
The binding sites 1n kidney appear to be of a single order of
affinity with an apparent binding constant (Kd, i.e. the concentration
of cyclic AMP needed to half saturate the binding sites) of 39 nM. The
binding
constant
1s
close
to
those
reported
for
several diverse
mammalian tissues with a single class of binding sites (Walton and
Garren, 1970; Montague and Howell, 1972; Sanborn et al., 1973; Talmadge
et al., 1975; Harbon et al., 1976; Marcus et al., 1979).
Dufau et al.
(1976) also described a single class of binding sites in Leydig cells,
although the binding affinity for cyclic AMP was 7 - 9 fold greater than
reported here.
Harbon et al. (1976) reported an intracellular binding
constant in myometrium consistent with that of Leydig cells, suggesting
that the reduced affinity results from in vitro preparation.
It 1s widely accepted that peptide hormones, such as PTH, initiate
some of their actions on target cells by binding to specific receptors
and resulting in the stimulation of adenylate cyclase.
tissues,
In these target
the cyclic Al.'1P generated appears to function as the major
second messenger to evoke hormone-specific biochemical responses (Sutherland and Rall, 1960; Robison et al., 1971).
function
to modulate
While cyclic AMP could
transcription of messenger
RNA,
through its
ability to stimulate RNA synthesis and phosphorlyation of nonhistone
chromatin proteins by cyclic AMP - dependent protein kinase (Allfrey et
30
al., 1973; Kish and Kleinsmith, 1974), most of the short-term actions of
cyclic
AMP
1n
extranuclear
eukaryotic
cells
phosphorlyation
of
are
believed
regulatory
to
be
proteins.
expressed
by
Considerable
evidence indicates that proteins exhibiting high binding affinity for
cyclic AMP are regulatory subunits of cyclic AMP - dependent protein
1970; Chambaut et al., 1971; Erlichman et al.,
kinases (Tao et al.,
1971; Garren et al., 1971; Reimann et al., 1971; Kumon et al., 1972).
Consequently, the binding of the cyclic nucleotide to the regulatory
subunit of the cyclic AMP - dependent protein kinases can be considered
as the first event occuring in the cell after the generation of cyclic
A1'1P
(Gill and Garren,
1970; Kumon et al.,
Reimann et al., 1971; Brostrom et al., 1971).
to
the regulatory subunit
regulatory
subunit.
subunit
-
in vitro,
cyclic
AMP
1970; Tao et al.,
Hhen cyclic A1'1P is bound
the enzyme dissociates
complex
1970;
and
an
active
into a
catalytic
The activation of protein kinases by cyclic AMP (cAMP) can be
described by the equation:
R-C
+
cAMP-=:=~
R-cAMP + C
(active)
(inactive holoenzyme)
The equation suggests that 'bound cyclic AMP' can be used as a direct
measure of the amount of active catalytic subunit.
Several studies have
demonstrated satisfactory correlations between intracellular cyclic
AMP levels and the active state of protein kinases in a variety of
intact tissue preparations
(Corbin et al.,
1973; Soderling et al.,
31
1973; Walaas et al., 1973; Korenman et al., 1974; Means et al., 1974;
Field et al., 1975; Keeley et al., 1975).
Therefore, the extent to
which cyclic AMP is bound to the regulatory subunit protein should be a
good indicator of the hormone - initiated activation of protein kinase
and subsequent hormone-specific biochemical responses.
PTH and PGE1 each stimulated cyclic AMP formation and increased
occupancy of cyclic AMP binding sites.
Hormone concentrations suf-
ficient to promote measurable increases in cyclic AMP significantly
depleted unoccupied binding sites (Figs. 2,3).
PTH-dependent binding
site depletion reached a plateau at a cyclic AMP concentration well
below the maximally achieveable level (Fig. 2). Maximal PGE1-dependent
binding site depletion was achieved without significant cyclic AMP
increase (Fig. 3).
were
employed
The lowest concentration of either PTH or PGE1 that
significantly
depleted
unoccupied
binding
sites.
A
similar dilemma has existed for the hCG-Leydig cell system; steroidogenesis was stimulated by hormone concentrations which produced no
perceptible increase 1n cyclic AMP (Mendelson et al., 1975; Moyle et
al., 1975; Sharma et al., 1976).
cyclic
k~P
receptor
analysis.
This dissociation was resolved by
Although increments
1.n cyclic AMP
content ,.,ere not detected after incubation with steroidogenic concentrations of hCG, a major reduction in unoccupied cyclic AMP receptor
content gave unequivocal evidence for increased cyclic AMP production
(Mobius et al., 1970).
The results from kidney are consistent with
those reported for Leydig cells and since binding to protein kinase
constitutes the primary,
if not sole,
action of cyclic AMP,
these
32
results support the concept that fluctuations in physiological concentrations of hormone can regulate cellular activity by inducing only
modest changes in intracellular cyclic AMP.
Activity of PTH and PGE1 on cyclic AL'1P formation as evidence
indicative of the presence of distinct target cells to each hormone in
renal cortex (Nagata et al.,
hormones can act
~n
additive
1978).
However, a finding that two
fashion
~n
a given tissue does
not
demonstrate target cell heterogenity, but rather may mean that receptors exist in fewer numbers than the cyclase molecules in the particular
group of target cells (Cuatrecasas et al., 1975).
The fact that PTH and
PGE1 share a common target, at least partly, in cortex might be related
to the similarity of their actions in regulating renal electrolyte
metabolism in rat kidney.
however,
doubt.
the physiologic
In view of the results just discussed,
importance of such additivity remains
~n
Since no further depletion of cyclic AMP binding sites occured
when PTH was added to maximally-effective PGE1, one might predict that
the more distal events in hormone action should also not be enhanced by
combining these agonists.
Elevated cyclic AMP levels induced by MIX or theophylline are
accompanied by increased occupancy of cyclic AMP binding sites (Table
3). To a given concentration of cyclic AMP, obtained in response to
either PGE1, MIX or theophylline, identical degrees of binding site
depletion were found.
Moreover, mode of cyclic AL'1P elevation does not
appear to modify the apparent affinity of the receptor for the cyclic
33
nucleotide.
Hence, it appears that neither hormonal stimulation nor
cessation of cyclic AMP degredation affect the intracellular equilibrium of free and bound
cyclic~~
(Harbon et al., 1976).
Maximal depletion of unoccupied binding sites by PTH or PGE1 led to
residual binding of approximately 60% of basal
levels.
Residual
binding of this magnitude has also been observed in other tissues
(Harbon et al., 1976; Marcus et al., 1979).
as
evidence
for
target
This has been interpreted
cell heterogeneity,
1.. e.
the binding site
depletion observed is the aggregate of cells which respond and do not
respond to specific agonists or groups of agonists (Marcus et al.,
1979).
Results of treatment with phosphodiesterase inhibitors seem to
conflict with this
interpretation,
assuming
identical permeability
between cell populations, since the resultant residual binding was like
that observed with hormone treatment.
Another interpretation for the
residual binding is that a portion of the binding sites in each cell
rema1.ns sequestered from newly-formed cyclic AMP.
This explanation
implies that the residual binding is undissociated holoenzyme.
cyclic
k~P-binding
Free
protein in freshly prepared tissue homogenates has
been reported, suggesting that the enzyme is partially dissociated in
v1.vo (Schubart and Rosen, 1976). Others have also reported the presence
of cyclic AMP-binding protein devoid of kinase activity (Chambaut et
al.,
1971; Jungmann et al.,
suggested
that
the
absence
1974).
of
free
Schubart and Rosen 0976) have
catalytic
subunit
in
freshly
prepared homogenates may be explained by preferential degredation of
catalytic subunit, which is relatively unstable in vitro, or that the
34
regulatory subunit could be produced in excess of the catalytic subunit
~n
v~vo.
Whether the excess cyclic AMP receptors are produced in excess
or they result from preferential degradation, it seems plausible that
their function might be to increase sensitivity of the target cell to
biologically significant levels of hormone by providing an increased
population of receptors to maintain the binding equilibrium.
Estimation of total cyclic AMP binding capacity of renal cortex
cytosol by isotope exchange assay showed that unoccupied sites accounted for 50% of the total unstimulated binding capacity (Table 1).
Similar results have been reported for diaphragm, myometrium, Leydig
cells and bone cells (Do Khac et al., 1973; Harbon et al., 1976; Dufau
et al., 1977; Marcus et al., 1979).
Depletion of unoccupied binding
sites by PTH did not significantly reduce the total binding site number
(Table 1).
Thus, binding of the regulatory subunit to cyclic AMP did
not lead to destruction or removal of the binding protein. In addition,
restoration of unoccupied binding sites was not afected by cycloheximide (Fig. 5).
Therefore, short-term binding site
restoration
primarily involves recycling of regulatory subunit after discharge of
cyclic AMP, and does not require de novo protein synthesis. The fate of
liberated free nucleotide is uncertain.
Presumably, the major portion
is hydrolyzed by phosphodiesterase or is transported from the cell.
However, studies with ovarian granulosa cells open the possibility that
translocation
of
cyclic
(Jungmann et al., 1974).
&'1P
to
nuclear
acceptor
sites may occur
35
Considerable
interest
has
focused
on
the
loss
of end-organ
responsiveness which follows exposure to trophic hormones.
elementary form,
cupancy,
In its most
such agonist-specific desensitization reflects oc-
destruction or removal of cell-surface receptors
hormone (Mukherjee et al., 1975).
for
the
However, deseneitization of Leydig
cells to hCG follows application of hormone at levels insufficient to
occupy a major
1977).
fraction of available receptors (Tsuruhara et al.,
Thus, it appears that end-organ desensitization
~s
a complex
process which may develop at multiple levels between the initial
hormone-receptor interaction and the ultimate physiologic response.
Time course studies of PTH action showed that unoccupied binding sites
mirrored changes in cyclic AMP content; with restoration of basal
levels occuring by 45 minutes after hormone addition (Fig. 5).
In bone
cells, secondary administration of PTH was attended by blunting of both
the cyclic AMP response and binding site depletion while PGE1 or
calcitonin given as the secondary agonist briskly elevated cyclic AMP
and depleted binding sites (Marcus et al., 1979). Thus, depletion of
binding sites is apparently a direct consequence of elevating cyclic
AMP
concentration,
receptor regulation.
and appears
to be
independent of cell-surface
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