Download Natriuretic Factor

NEWS
IN PHYSIOLOGICAL
SCIENCES
ocytes.
The ANF
comes
from
these
granules, as demonstrated
by ultracentrifugation
studies, the unlabeled
antibody
technique
of Sternberger,
and the protein A gold technique (1).
The granularity
of these cells is
Natriuretic Factor
greater
Jacques Genest and Marc Cantin
right atrium when compared with
the left atrium and is more marked
major
role in vasorelaxation
and in hypertensive
and edematous
states.
The progress made in the last two years is a testimony to the powers of
the new analytical techniques for isolation and characterization of
peptides.
The regulation
of body fluid volumes
has long been a puzzle.
Bold
For example,
et al. (3) in 1981 of a rapid,
massive,
and short
diuresis
and na-
what factors are involved in correcting the volume expansion during
triuresis in rats following the intravenous administration
of rat atria1
and following
homogenates.
the intravenous
infu-
sion \ of isotonic saline? In other
words, how is the blood volume
Since this key observation,
progress has been spectacular (1). It is a
maintained
and the “milieu
interieur” kept within narrow limits?
magnificent
recent
discoveries
atria1 natriuretic
in heart atria contain
progress
standing
transfer
was made in our underof its physiology.
Gene
has already
which
in
are our own group at the Clin-
of Needleman,
expanded blood volume. The work
done on the granular
cells since
Louis; Atlas, Lewicki
York and California;
early
from Vanderbilt
major
groups,
Cantin in Montreal,
Bencosme in
Kingston, Canada, and Hatt in Paris,
has led to the demonstration
by de
]acques Genest is Consultant
and Marc Cantin
is Director,
Multidisciplinary
Research
Group
on Hypertension,
Clinical
Research
Institute
of Montreal,
110 Pine Ave. West, Montreal,
Quebec HZW lR7, Canada.
0886- 17 14/86 S 1.50 0 1986. Int. Union
Physiol.
Scl/Am.
Physiol.
been achieved.
ical Research Institute of Montreal,
directed by Marc Cantin; the groups
response to stretch, whether
caused
by increased
perfusion
pressure
or
1970 by three
was
This was due to the independent
efforts of several groups, among
gran-
contents
(ANF)
and the ANF gene in mouse, rat, and
humans
were
cloned,
and great
clarify
secretory
factor
purified,
sequenced,
and synthesized. The cDNA encoding for ANF
the major endocrine connection between heart and kidney. Many cells
ules that release their
of the pow-
ers of modern analytical tools in peptide chemistry and of molecular biology. Within less than 18 mo the
Several factors such as aldosterone, the antidiuretic
hormone arginine vasopressin,
and others play a
role in the regulation
of blood volume. The low pressure elements of
the heart, the atria, have more recently been implicated
as being in
the most suitable area to “sense the
fullness of the blood stream.”
Exciting
illustration
Sot.
Currie
et al. in St.
et al. from New
Inagami et al.
University;
Napier,
in the appendages
of Z-2.5
in the
of the atria
under the subpericardial
As demonstrated
first
and
surface (2).
in 1976 by
Marie, Guillemot,
and Hatt (7) and
later confirmed
in 1979 by de Bold
et al., the granularity
of these cardi-
ocytes varies greatly with the sodium and water balance.
Since June 1983, several groups
have isolated from homogenates
of
atria from humans,
and rats, natriuretic
in length
pigs, monkeys,
factors varying
from 21 to 33 amino acids,
all having in common a core of 21
amino acids with a cysteine-disulfide bridge essential for their activ-
ity. The exact length of the circulating ANF is not yet known, but the
evidence from cultures of atria1 car-
diocytes
liberated
indicates that the peptide
in the medium
is the Arg
lOl-Tyr
126 (22 amino acids), The
human atria1 natriuretic peptide has
28 amino acids with a sequence similar in all respects to the rat factor,
except for the presence of a methionine in position 110 instead of isoleucine. The amino acid sequence of
the ANF peptide is novel and could
not be found in any reported peptide
sequence.
Effect of ANF on renal
excretion of water and sodium
Present evidence suggests that the
diuretic.
and natriuretic
actions of
Nutt et al. from Rahway, NJ; Seidman et al. from Boston; Kangawa et
ANF are mainly due to its vasorelaxing effect on renal arteries and arte-
al. from
rioles and to a significant increase in
Japan; and Forsmann
et al.
from Heidelberg.
Most cells in the atria of all mam-
glomerular
filtration rate and filtration fraction, leading to an increased
mals, including
humans,
contain
specific granules at the poles of the
nucleus
as well as myofibrils,
and
these cells have been named cardi-
excreted fraction of filtered sodium.
Kaliuresis also occurs. This is
strongly supported by the findings of
Cantin et al., who showed by autoVolume
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1986
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The discovery of the atria1 natriuretic factor is a major breakthrough for
our understanding of the regulation of body fluid volume. It plays a
by a factor
How ANF affects
hypertension
experimental
Intravenous
administration
of
ANF as a bolus (1 pg) to rats with
Goldblatt
experimental
hypertension results in a lowering of systolic
blood pressure below 130 mmHg for
a mean duration
of 34-40 min.
(Goldblatt
hypertension
is induced
by a clip producing stenosis on one
renal artery in rats with intact kidneys or with one kidney removed,
designated Z-kidney, l-clip or l-kidney, l-clip rat, respectively.) Continuous intravenous
infusion at 0.1-l
pg/h for 6 days is accompanied
by a
significant fall of blood pressure to
normal levels after the 3rd day of
infusion and during the whole period of administration.
The effect is
less marked in the l-kidney,
l-clip
rats receiving
the 0.1,pg infusion
rate (4).
This antihypertensive
effect of
ANF appears more dependent on its
vasorelaxing effect than on the loss
of sodium and the subsequent decrease in extracellular
fluid volume.
Addition of synthetic ANF to a bath
4
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1/February
1986
containing
vascular strips from rat
and rabbit renal arteries and aorta
shows a profound inhibition
of the
contraction induced by norepinephrine and by angiotensin II. This vasorelaxation
is followed by a prolonged period of refractoriness to the
vasoconstrictor
effect of both agents.
Could the natriuretic
effect
of ANF act via aldosterone
secretion?
Probably not. The in vitro effects
of synthetic ANF on the production
of aldosterone in cell cultures from
the bovine adrenal zona glomerulosa
demonstrate
a 20% decrease in the
basal production
levels and a 4O70% inhibition
of aldosterone
production
following
stimulation
by
ACTH and by angiotensin II. A similar decrease in plasma aldosterone
levels has been shown in vivo during
infusions of ANF to rats.
The effect of ANF on aldosterone
secretion and plasma concentration
may have little relationship
with its
diuretic and natriuretic
effects because of the rapid and brief action of
ANF (onset within 2 or 3 min and
lasting only 15-20 min); in contrast,
the renal response to aldosterone is
apparent only after a delay of 45-60
min.
Effect of ANF
vasopressin
on arginine
Plasma arginine vasopressin (antidiuretic
hormone)
concentration
stimulated
by hemorrhage
or dehydration decreases markedly
during
the intravenous
administration
of
ANF. Thus a decrease in arginine
vasopressin may contribute
to the
diuresis. Specific binding sites have
also been demonstrated
in membranes of posterior pituitary cells.
Receptors
studies
Specific binding
sites for ANF
were characterized
by displacement
of the labeled ANF by “cold” ANF at
concentrations
of 10ml* M, but not by
other active peptides such as ACTH,
angiotensin II, arginine vasopressin,
insulin,
substance P, and others.
Specific tissue binding of ANF has
been demonstrated
in renal glomeruli, adrenal zona glomerulosa,
posterior pituitary,
and vascular endothelium. More recently three different groups from Montreal, Bethesda,
and St. Louis have shown specific
binding
sites by autoradiography
immunofluorescence
and
techniques in the rat brain, especially in
areas such as the area postrema, the
nucleus
tractus
solitarius,
the
subfornical
organ, and the anteroventral
third
ventricle
region
(AV3V), all areas involved in the regulation
of water and/or of blood
pressure.
Role of ANF in diuresis
and
natiuresis
following
volume
expansion
Both diuresis and natriuresis induced during intravenous
infusion
of isotonic saline in rats are markedly inhibited
by concomitant
administration
of ANF
antibodies
raised in rabbits against synthetic
ANF (Arg ?Ol-Tyr 126). This suggests that the diuresis is ANF induced.
Effects on adenylate
and cGMP
cyclase
ANF at concentrations
of lo-‘* to
lo-’ M inhibits significantly
the activity of adenylate
cyclase, especially when stimulated by isoproterenol, norepinephrine,
or glucagon in
purified preparations
of mesenteric
artery homogenates,
aorta washed
particles,
and renal homogenates.
Administration
of synthetic ANF in
rats or in renal cell cultures in vitro
is accompanied by a very important
rise in plasma levels and urinary excretion
of guanosine
3’,5’-cyclic
monophosphate
(cGMP), which appears to be a marker for the action
of ANF. In aortic strips, the increase
in cGMP parallels the vasorelaxation.
Does ANF affect the sodium
transport
system?
No effect could be demonstrated
on the Na’-K+-ATPase
activity, the
Na+-Li+ countertransport,
and the
Na’-K+ co-transport
system in red
blood cells or in renal, myocardial,
and vascular smooth muscle cells.
Direct
immunoassay
of ANF
Several groups have succeeded in
establishing a sensitive and specific
radioimmunoassay
for the measurement of the ANF, not only in atria1
tissues but also in plasma of rats,
dogs, and humans. Administration
of
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radiography an almost exclusive localization of labeled ANF in the glomeruli, renal arteries, and arterioles
and its absence along the proximal
and distal tubules and collecting
ducts.
In receptor studies by De Lean et
al., there was very intense specific
binding of labeled ANF over the glomeruli, as well as slight binding over
the distal tubules and collecting
ducts. Other workers have shown
that, with low doses of ANF, significant diuresis and natriuresis can occur without any change in glomerular filtration
rate. This suggests a
tubular effect. But there are divergent opinions as to whether the ANF
acts through a decrease in sodium
reabsorption along the proximal tubule (since ANF administration
also
increases the excreted fraction of filtered lithium
and phosphates) or
along the distal tubule and collecting
ducts, as suggested by the work of
others. The role of ANF on the renal
tubular transport of sodium remains
to be elucidated.
The presence of
Asn-122, Ser-123, and Phe-124 at the
carboxyl terminal is essential for the
diuretic, natriuretic,
and vasorelaxing activity of ANF (5).
coronary sinus blood is two to seven
times higher than that found in
blood of the right atrium and in peripheral
venous or arterial blood.
There is therefore direct evidence
that both in humans and in rats the
atria1 natriuretic
factor elaborated
and localized in the granules of the
atria1 cardiocytes is released in blood
and is therefore a true circulating
hormone (6).
This work
was supported
R esearch Cou ncil of Canada.
by the
Medical
References
1. Cantin,
M., and J. Genest.
The heart and
the atria1 natriuretic
factor.
Endocrine
Rev. In press.
2. Chapeau,
C., J. Gutkowska,
P. W. Schiller,
R. W. Milne,
G. Thibault,
R. Garcia,
J.
Genest,
and M. Cantin.
Localization
of
immunoreactive
atria1 natriuretic
factor
(ANF) in the heart of various
animal species. J. Histochem.
Cytochem.
In press.
3. De Bold A. J., H. B. Borenstein,
A. T Veress, and H. Sonnenberg.
A rapid and po-
The Patch-Clamp Technique:
Recording Ionic Currents
Through Single Pores in the
Cell Membrane
0. H.PetersenandC.C.H.Petersen
When an intracellular
electrode records an electrical event in the cell
membrane,
we know that there are changes in the membrane’s
permeability
to various ions. These changes are mediated
by
membrane
entities known as pores or ion channels. The advance of the
patch-clamp
technique,
which permits the study of changes in
individual
ion channels,
in the simplest case conforms to a simple
open-close two-state model.
brane current flow. The influence of
neurotransmitters
or of electrical
stimuli
are described in terms of
changes in membrane
conductance
or membrane
permeability
to various ions. While this approach has
been enormously
successful in defining and accounting for the macroscopic transmembrane
ionic currents associated with, for example,
the nerve action potential (4), it is by
Electrophysiological
investigations
emoloving
intracellular
electrodes
tre& the-plasma
membrane
as a
black box. Measurements
are made
of the transmembrane
electriqal potential difference or of transmem0. H. Petersen and C. C. H. Petersen are in the
Medical
Research
Council
Secretory
Control
Research
Group,
Department
of Physiology,
University
ofLiverpool,
PO Box 147, Brownlow
Hill, Liverpool
L69 3BX, United
Kingdom.
0886- 17 14/86 $1.50 0 1986 Int. Union
Physlol.
Scl/Am. Physiol.
Sot
tent natriuretic
response
to intravenous
injection
of atria1 myocardial
extract
in
rats. Life Sci. 28: 89-94,
1981.
Garcia,
R., G. Thibault,
J. Gutkowska,
P.
Hamet, M. Cantin,
and J. Genest. Effect of
chronic
infusion
of synthetic
atria1 natriuretic factor (ANF 8-33) in conscious
2KK hypertensive
rats. Proc. Sot. Exp. Biol.
Med. 178: 155-159,
1985.
Garcia,
R., G. Thibault,
N. G. Seidah, C.
Lazure,
M. Cantin,
J. Genest,
and M.
Chretien.
Structure
activity
relationships
of atria1 natriuretic
factor
(ANF).
Biohem.
Biophys.
Res. Commun.
125: 938946,
1984.
Gutkowska,
J., M. Bourassa,
D. Roy, G.
Thibault,
R. Garcia,
M. Cantin,
and J.
Genest.
Immunoreactive
atria1
natriuretic factor (IR-ANF)
in human
plasma.
Biochem.
Biophys.
Res. Commun.
In
press.
Marie,
J. P., H. Guillemot,
and P. Y. Hatt.
Le degre de granulation
des cardiocytes
auriculaires.
Etude
planimetrique
au
tours des differents
apports
d’eau et de
sodium
chez le rat (Abstract
in English).
Pathof. BioJ. Paris 24: 524-554,
1976.
its very nature incapable of giving
direct information
about the underlying microscopic events.
How to detect single-channel
currents
Downloaded from http://physiologyonline.physiology.org/ by 10.220.33.3 on May 11, 2017
ether or morphine,
as well as immobilization
stress for 4 h in rats,
stimulates a 5- to ZO-fold increase of
immunoreactive
ANF (IR-ANF) in
plasma when compared with values
obtained
in conscious
rats. The
plasma concentration
of IR-ANF is
increased ninefold in hamsters with
hereditary cardiomyopathy
and congestive heart failure compared with
control hamsters. In these animals
with congestive heart failure, the
atria1 cardiocytes are hypertrophied
with a tremendous
increase in the
size of the Golgi complex and a reduced number of granules.
Administration
of ANF to normal
human subjects results in marked
increases in urine volume and in
natriuresis accompanied by a significant decrease in both systolic and
diastolic pressures. Blood obtained
from the coronary sinus in patients
with various heart conditions shows
that the concentration
of IR-ANF in
Electrical currents in cells are mediated by a single class of proteins in
the plasma membrane
called ion
channels or pores. The first direct
observation
of current
flowing
through single-ion
channels was a
result of studies in bimolecular
lipid
membranes in the presence of antibiotics (3). In intact cells the problem
is to detect single-channel
currents
in the presence of background electrical noise. Conventional
intracellular microelectrode
methods
for
current measurement
are associated
with a background noise of at least
100 pA, whereas the current flowing
when a single channel opens is only
a small fraction of this background
noise.
Neher and Sakmann (7) solved this
problem
by the
patch-clamp
method. Instead of inserting a microelectrode into a cell they pressed a
microelectrode
tip onto the surface
of a cell, effectively isolating a patch
of membrane. The intrinsic noise increases with the area of the memVolume 1/February
1986
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