Note: Descriptions are shown in the official language in which they were submitted.
1340622
OBJECT OF THE INVENTION
This invention relates to a diuretic and natriuretic
factor and a process for extraction thereof from mammalian
heart atria, especially rat hearts.
BACKGROUND OF THE INVENTION
It is known that the muscle cells of the atrial
myocardium in mammals contain, in addition to contractile
elements similar to those found in ventricular fibers, a
highly developed Golgi complex, a relatively high propor-
tion of rough endoplasmic reticulum, and numerous membrane
bound storage granules, referred to as specific atrial
granules. No such granules appear to exist in ventricular
muscle cells. Morphologically and histochemically the
atrial granules resemble those present in polypeptide-hormone
producing cells (J. Histochem. Cytochem. 26, 1094 - 1102
(1978) de Bold et al). It is also known (Life Sciences,
Vol. 28 pp 89-94 (1981) de Bold et al) that injection of a
crude extract of rat atrial myocardium induces a very
potent and immediate natriuretic response in non-diuretic
assay rats. The problem remains, however, to isolate and
identify the atrial natriuretic factor (ANF) contained
in the crude extract of the specific atrial granules,
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1340622
and which is hereinafter referred to as cardionatrin I.
STATEhIENT OF INVENTION
It is, therefore, an object of the present
invention to provide a process for the extraction of ANF
from mammalian heart atria.
It is another object of the invention to provide
a peptide extract from mammalian heart atria having
diuretic and natriuretic activity.
Thus, by one aspect of the invention there is
provided a process for extracting a diuretic and natriuretic
factor from mammalian heart atria comprising:
(a) homogenizing mammalian heart atria in an
aqueous solution containing a lower carboxylic acid to
thereby extract said factor into said solution;
(b) adjusting the pH of said solution within
the range 4.5 to 7.6 to thereby precipitate impurities
therefrom; and
(c) chromatographically purifying said natriuretic
factor remaining in said solution.
By another aspect of the invention there is
provided a diuretic and natriuretic peptide composition
obtained by carboxylic acid extraction from mammalian
heart atria or synthetically, comprising a derived 28
amino acid sequence.
DESCRIPTION OF DRAWINGS
The invention will be described in more detail
hereinafter with reference to the following drawings in
which:
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1340622
Figure 1 is a graph illustrating chromatographic
desalting of rat atrial extract in Bio-Gel~ P-2. Sample
size: 10 ml of acetic acid extract obtained from 5 g of
tissue. Column size: 2.6 x 60 cm. Fraction Size: 11 ml.
Eluant: 1.0 M acetic acid. Linear flow rate: 2 cm/h.
Hatched area indicates fractions where natriuretic activity
is recovered;
Figure 2 is a graph illustrating chromatography
of rat atrial extract (after desalting in Bio-Geln P-2)
in Sephadex~ G-75. Column size 2.6 x 90 cm. Fraction
size 11 ml. Eluant: 1.0 M acetic acid. Linear flow rate:
5 cm/h. Natriuretic activity (bars) was determined by
injecting freeze-dried aliquots of pooled fractions
resuspended in PBS;
Figure 3 is a bar graph illustrating typical effect
of an injection of partially purified rat atrial natriuretic
factor on arterial blood pressure and urine output (in drops)
of bioassay rats;
Figure 4 is a graph illustrating net natriuretic
and diuretic effect of partially purified natriuretic
factor injected in a total volume of 0.2 ml of PBS. Net
values were obtained by subtracting total sodium excretions
and urine output of animals injected with PBS alone from
corresponding values of animals injected with natriuretic
factor. (Values ~ S.E.M.); and
Figure 5 is a bar graph illustrating ion exchange
chromatography purification of partially purified natriuretic
factor. Hatched area indicates fractions where natriuretic
activity is recovered.
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1340622
Figure 6 is an RP-HPLC elution profile of an
acid extract of rat atrial muscle.
Figure 7 is an RP-HPLC elution profile of region
I of Figure 6, after chromatography in HFBA-containing
CH3CN gradient;
Figures 8a and 8b are RP-HPLC elution profiles
obtained after rechromatography in a TFA-containing
CH3CN gradient of active material from chromatography using
HFBA (Figure 7);
Figure 9 is a graph of molecular weight versus
migrated distance for SDS-Page comparison; and
Figure 10 is a UV absorption spectrum of
cardionatrin I.
DETAILED DESCRIPTIOPJ OF PREFERRED EMBODIP~IENTS
The presence of secretory-like specific atrial
granules in cardiac atrial muscle fibers which has been
recognized for some years (J. Cell. Biol. 23, 151 (1964)
Jamieson et al) and the more recent finding that crude rat
heart atrial muscle extracts are able to induce a very
potent diuretic and natriuretic response in assay rats
(Life Sciences 28, 89, (1981) de Bold et al) is believed
to be of considerable importance to studies for in-vivo
water and electrolyte balance. The problem remains. however,
that the crude extract heretofore produced is not sufficiently
purified to make a positive identification of the active
factor. While a simple homogenization of heart tissue in
water or buffered solutions may be employed for extraction
of ANF, such a procedure is relatively slow and the
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134oszz
concentration of the resulting ANF is very low. Upgrading
is tedious and costly. Somewhat surprisingly, although
extractions from large mammalian hearts, such as beef,
can be effected the natriuretic factor therein appears
to be but a very minor component even in the active U.V.-
absorbing material isolated after ion exchange chromato-
graphy and the total extractable activity appears to be
much lower than that obtained from an equivalent amount
of rat tissue. Rat atria are therefore the preferred
source material.
It has now been determined that advantage may be
taken of the well known peptide-solvent properties of
lower carboxylic acids such as formic and acetic acid
etc., and two alternative techniques will be described
in more detail hereinafter. In both techniques, rat atria
provided the source material. In the first technique
use is made of the extractive properties of 1.0 M acetic
or other lower carboxylic acid optionally containing
protease inhibitors. Following extraction, some contaminants
are elminated by precipitation after adjusting the pH to
a range between about 4.5 to about 7.6. The volume of the
extract is then reduced, preferably by freeze drying and
further purified by gel chromatography and ion exchange
chromatography.
Example 1
Atria were obtained from male Sprague-Dawley rats
(300-350 g). Up to 100 atria (10 g wet weight) were used
in each experiment. The animals had free access to food
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13~OG~~
and water until sacrificed by decapitation. The hearts
were rapidly removed and placed in ice cold phosphate
buffered saline (PBS: 0.9~ NaCl in 5 mM sodium phosphate
buffer, pH 7.2). The atria were dissected, thoroughly
rinsed in PBS, blotted dry and weighed. Ventricular
tissues were similarly treated to provide controls. Five
volumes of cold 1.0 M acetic acid, containing 1 mg of the
protease inhibitors pepstatin A and phenylmethylsulfomyl
fluoride, were added per gram of tissue wet weight. The
tissue was then homogenized in a Polytron~, let to stand
for 1 h on ice and centrifuged at 4°C for 30 min at 10,000 g.
The supernatant from this centrifugation was saved and the
pellet re-homogenized in 2.5 volumes of acetic acid and
treated as above. The combined supernatant from the two
acetic acid extractions was then adjusted to pH 7.6 and
centrifuged once again. The supernatant from this
centrifugation was freeze-dried, resuspended in 1.0 M
acetic acid and desalted at 4°C in a Bio-Gel~ P-2 column
(2.6 x 60 cm) equilibrated with 1.0 M acetic acid. The
void volume from this column was freeze-dried, resuspended
in 1.0 M acetic acid and applied to a Sephadex~ G-75
column (2.6 x 90 cm) equilibrated with 1.0 M acetic acid.
Pooled active fractions obtained after this chromatography
were freeze-dried. The product obtained is referred to as
"partially purified natriuretic factor".
Example 2
To study the effect of protease (Pronase) on
partially purified natriuretic factor, dilutions were made
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134oszz
in 0.05 M TRIS buffer, pH 7.6. Digestions were carried
out by incubating 50 a g of partially purified natriuretic
factor with 0.1 U of Pronase for 4 h at 37°C in a total
volume of 1.1 ml of 0.05 M TRIS buffer, pH 7.6. The
reaction was terminated by addition of 0.2 ml of glacial
acetic acid and cooled on ice. After freeze-drying, the
samples were resuspended in 1.0 ml of PBS, centrifuged,
and 0.2 ml aliquots tested for natriuretic activity.
Further purification of natriuretic factor was
accomplished by ion exchange chromatography in CM Bio-Gel~
equilibrated with 0.1 M ammonium acetate buffer, pH 4.7.
Column size was 0.9 x 12 cm. Elution was carried out
with 13 column volumes under starting conditions followed
by a 13 column volumes gradient to 0.1 M ammonium acetate,
pH 7.0 and, finally, with 13 column volumes from 0.1 M
ammonium acetate, pH 7.0 to 0.5 M ammonium acetate, pH 7Ø
Example 3
Natriuretic factor assays were carried out in
non-diuretic rats as follows:
Male Sprague-Dawley rats (wt. range 250-372 g)
were anaesthetized (Inactin~, 10 mg/100 g body wt. i.p.)
and prepared for bioassay. Arterial blood pressure and
heart rate were measured through a femoral artery cannula.
A bladder catheter allowed quantitative collection of
urine, and a femoral vein cannula was used for main-
tenance infusion and injection of test material. On
completion of surgery a priming dose of Ringer's solution
(1.2 ml) containing 3H inulin (4.0 uCi/ml) was
- 7 _
l~4oszz
administered over 20 min, followed by constant infusion
of the same solution throughout the experiment at 1.2
ml/hr. The animals were let to stabilize for a further
20 min. During the following 20 min urine was quanti-
tatively collected (control period) after which injection
of 0.2 ml of the test sample was made over approximately
5 s. Urine was then collected for the next 20 min
(test period). A photoelectric drop counter was
positioned between the bladder catheter and the collection
tube to qualitatively assess the diuretic response. Urine
sodium and potassium concentrations were measured by
flame photometry, chloride by electrometric titration
and urine volumes by weighing. Protein estimation was
made by measuring absorbance at 280 nm (10 mm path)
assuming lAU - 1 mg protein/ml. Statistical comparisons
were carried out using unpaired student's t-test.
Results
Pooled active freeze-dried fractions, obtained
after chromatographic desalting in Bio-Gel~ P-2 (Figure 1)
or after fractionation in Sephadex~ G-75 (Figure 2), and
injected dissolved in PBS, induced a typical response in
assay rats (Figure 3). In terms of urine output, this
response was characterized by rapid onset (1-2 min) and
decay (10-15 min). By the end of the test period -
i.e. 20 min after injection - urine output was essentially
the same as that observed during the control period.
Doses approaching maximal response had the effect of
gradually decreasing arterial blood pressure so that
_ g _
134062
the blood pressure values observed at the end of the
assay were 10-30 mm Hg lower than values observed prior
to the injection of the extracts.
Both diuretic and natriuretic responses were
dose dependent (Figure 4). Maximal response obtained
after injection of 18 ug of partially purified factor
corresponded to a 30-40 fold and 10-15 fold increase in
total sodium excretion and urine volume respectively.
Incubation of partially purified natriuretic
factor with protease is illustrated in Table I overleaf,
and shows completely abolished activity while samples
incubated with inactivated enzyme had activities compar-
able to that of samples incubated with buffer alone.
Protease digestion was carried out by incubating
approximately 50 ug of partially purified natriuretic
factor with 0.1 U of protease for 4 h at 37°C in a total
volume of 1.1 ml of 0.05M TRIS buffer, pH 7.6 (Group B).
Controls were incubated with either boiled protease
(Group C) or with buffer alone (Group A). (All values
~ S.E.M.)
_ g _
1340622
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1340622
Dlatriuretic factor distribution during
chromatography in Sephadex~ G-75 (Figure 2) was consis-
tently multimodal although most of the activity was
recovered in peaks corresponding to molecular weights
of less than 6,000 Daltons. Further purification of
this fraction was accomplished by ion exchange chroma-
tography in CM Bio-Gelo (Figure 5) after which natriuretic
activity was recovered in a single, though not symmetrical,
peak.
The fact that natriuretic activity is recovered
after desalting in a Bio-Gel<"? P-2 column (normal fraction-
ation range: 100-1,800 Daltons) is of considerable
interest because it shows that natriuretic factor is
distinct from inorganic salts and low molecular weight
organic compounds which are likely extracted by acetic
acid and which may be expected to be cardioactive and/or
affect kidney function.
The significance of multimodal distribution
of natriuretic factor after chromatography in Sephadex~
G-75 is not clear. Protein interaction, possibly including
polymerization, appears as the most likely explanation.
Protease sensitivity as well as the general
behaviour of natriuretic factor suggest that it is a
polypeptide.
The second technique for extraction of ANF
follows a general outline for the isolation and purifica-
tion of pituitary peptides described by Bennett et al
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1340622
"Purification of the Two Major Forms of Rat Pituitary
Corticotrophin Using Only Reverse-Phase Liquid Chroma-
tography" Biochemistry, 20: 4530-4558, (1981). Heart
tissues are homogenized in an aqueous extractant mixture
typically containing 1.0 M acetic acid, 1$ sodium chloride
and 1.0 M hydrochloric acid. Other organic acids, mineral
acids or salts may, of course, also be used. The extracts
are then treated with octadecylsilyl silica in a batch
procedure and ANF is further purified using reverse phase
high performance liquid chromatography.
Example 4
Rat atria, freshly dissected and frozen, were
obtained commercially. The frozen tissue was finely ground
and an acetone powder prepared by repeated extraction with
acetone and hexane. Up to 700 mg of the dried powder was
extracted three times with 20, 10 and 10 mL respectively
of an aqueous extractant consisting of 1.0 M acetic acid,
1.0 M hydrochloric acid and 1$ sodium chloride. Extraction
was carried out on ice using a ground-glass homogenizer.
The extracts so obtained were centrifuged and the
supernatant was passed through two octadecylsilyl silica
(ODS-silica) cartridges C18 Sep-Pak~, Waters Associates).
Before use, the cartridges were wetted with 5 ml of 80$
acetonitrile (ACN)/water containing 0.1~ trifluoroacetic
acid (TFA) and rinsed with 5 ml of 0.1$ TFA. After
passing the samples, the cartridges were rinsed with
20 ml of 0.1~ TFA. Compounds bound to the cartridges
(including atrial natriuretic factor) were eluted by
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1340622
passing 3 ml of 80$ ACN/0.1~ TFA. The eluate from each
cartridge was diluted to 18 mL with 0.1~ TFA and pumped
through the "aqueous" pump of a high performance liquid
chromatograph for binding to a u-Bondapako C18 chromato-
graphic column (Waters Associates) previously equilibrated
with 12$ ACD1/0.1~ TFA. The column was then eluted using
a gradient of 20 to 40$ ACN. Two active fractions are
recovered. These fractions were further purified by
separate rechromatography in the same system this time
using ACN gradients containing 0.1~ heptafluorobutyric
acid (HFBA). Active fractions from HFBA gradients were
purified once more by another gradient chromatography
using TFA-containing ACN gradients. Injection of test
samples of the extract thus produced into non-diuretic
rats according to the procedure outlined in Example 3
resulted in similar results to those described in Example 3.
Example 5
Atria were obtained from adult male Sprague-
Dawley rats. The tissues were kept frozen (-70°C) from
24 to 72 h before being pulverized in a household-type
coffee grinder previously cooled by grinding dry ice
chips. The frozen tissue powder was then poured into
a beaker containing 10 volumes of an ice-cold aqueous
extractant composed of 1.0 M acetic acid, 1.0 N HCL and
1$ NaCl and homogenized using a Polytron fitted with a
PT35 probe operated for 60 s at 50~ power. The homogenate
was stirred for 1 h in the cold before centrifuging. The
pellets thus obtained were re-extracted following an
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1340622
identical protocol but using 5 volumes of the extractant.
The supernatants from the two extractions were combined
and 40 ml aliquots were passed five times through Sep-Pak~
cartridges (Waters) which were then washed with 20 ml
of 0.1$ trifluoracetic acid (TFA) and eluted with 3 ml
of 80~ acetonitrile (CH3CN) in 0.1~ TFA. The eluates
were then diluted with 0.1$ TFA in a proportion of 15 ml
of acid to 3 ml of cartridge eluate. The diluted eluate
was pumped into a HPLC column (u-Bondapak~ C18, 7.8 x
300 mm) through the "B" (aqueous) pump of a series 2/2
Perkin-Elmer~liquid chromatograph. The column had
previously be equilibrated with 12$ CH3CN in 0.1~ TFA
and gradient elution was carried out over 75 min at
1.5 ml/min with a linear gradient of 20 - 50~ CH3CN in
0.1~ TFA. Column effluent was monitored at 278 nm
(LC55 Perkin-Elmer~detector) and collected in 2 min
fractions. Aliquots of the fractions were freeze-dried
and re-suspended in phosphate-buffered saline (PBS) for
natriuretic factor assay. Figure 6 shows the elution
profile of extracts obtained from 200 atria after RP-HPLC.
The extract obtained from 200 rat atria was pumped directly
into the chromatographic column (u-Bondapak<"~ C18, 7.8 x
300 mm) and eluted over 75 min with a gradient of 20-50~
CH3CN in 0.1~ TFA. Two min fractions were collected.
Aliquots of these fractions were freeze-dried and re-
suspended in PBS for assay of natriuretic activity.
Activity was found distributed in four discrete regions
of the chromatogram. The fractions comprising region I
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1340622
were pooled, diluted 1:1 with aqueous 0.13$ hepta-
fluorobutyric acid (HFBA) and pumped back into the
column which had been equilibrated with 12~ CH3CN in
0.13$ HFBA. Elution of the column was carried out over
40 min with a gradient of 28 - 44~ CH3CN in 0.138 HFBA.
Activity was recovered in fractions eluting between 31
and 34 min (Fig. 7). These fractions were diluted l:l
with 0.1$ aqueous TFA and re-chromatographed using a
3.9 x 300 mm N-Bondapak~ C18 column eluted over 20 min
with a gradient of 20 - 36$ CH3CN in 0.1$ TFA. As shown
in Figure 8, a sharp, symmetrical peak was now obtained
which was apparently homogeneous at both 278 nm (Figure 8a)
and 215 nm (Figure 8b).
The product obtained at this point is herein-
after referred to as "cardionatrin I". Total yield of
cardionatrin I varied from 12 - 20 nmol per 1,000 atria.
The material obtained from 200 rat atria was used to
obtain a molecular weight estimate using area-sodium
dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).
Further characterization studies on "cardio-
natrin I" have been carried out using preparations of
cardionatrin I derived from 1,000 rat atria, as follows:
amino acid analysis after hydrolysis in 6 td HCL both
with or without prior performic acid oxidation; amino
acid analysis after hydrolysis in 4 N methanesulfonic
acid; spectrophotometric determination of tryptophan.
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1340622
The molecular weight of cardionatrin I was
estimated by SDS-PAGE. Cardionatrin I (I) obtained
from a 200-rat atrial extract, was analyzed by urea
SDS-PAGE together with molecular weight standards. The
standards were cyanogen bromide fragments of sperm whale
myoglobin (MF) and commercially obtained glucagon (G).
Plot of molecular weight versus migrated distance
(Fig. 9) gives an estimate of 5,150 for the molecular
weight of cardionatrin I (I).
Amino acid analysis (Table II)
Table II. Amino Acid Composition of
Cardionatrin I
Amino Acid No. of Residues
Asp 3.7 (4)
Thr 1.3 (1)
Ser 6.4 (6)
Glx 4.8 (5)
Pro 1.9 (2)
Gly 7.3 (7)
Ala 3.3 (3)
Cys 0.9 (1)
Val 1.0 (1)
Met 0.8 (1)
Leu 3 . 2 ( 3 )
Tyr 1.1 (1)
Phe 1.8 (2)
Lys 2.8 (3)
His 1.1 (1)
Trp 0 . 0 ( 0 )
Arg 5.7 (6)
Total no.
of residues 47
revealed that the peptide consisted of 47 residues one
of which was cystine indicating that cardionatrin I
contains a disulfide bond. No trytophan residue was
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1340622
detected by amino acid analysis following hydrolysis
of the peptide in 4 N methanesulfonic acid. The UV
spectrum of the peptide also did not show the shoulder
at 288 nm characteristic of tryptophan containing
peptides (Fig. 10). The spectrum was obtained using a
Caryo210 spectrophotometer with a 10 mm light path and
equipped with automatic baseline adjust. For the
spectrum shown, 10 nmol of cardionatrin I were dissolved
in 1.0 mL of 0.1 N HCL. The molecular weight of
cardionatrin I estimated from the amino acid composition
was 5273 which is in close agreement with that estimated
by SDS-PAGE gel electrophoresis.
Injection of 0.5 nmol of purified cardionatrin
I induced a characteristic diuretic response of rapid
onset and decay in the non-diuretic bioassay rat,
resulting in a two-fold increase in urine output and a
four-fold increase in sodium excretion. From previous
dose-response studies using partially purified prepara-
tions, maximal bioassay response for cardionatrin I may
be expected to be in the 1 to 2 nmol range.
Experiments to determine the sequence of the
amino acids in the peptide described above, or at least
that portion of the cardionatrin I which exhibits
biological activity, i.e. diuretic properties, have
R
been conducted using a Beckma~890C automatic sequencer.
Edman degradation followed by analysis of phenylthio-
hydantoin amino-acids by high pressure liquid
chromatography resulted in a derived sequence as follows:
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1340622
Serine - Leucine - Arginine - Arginine - Serine -
Serine - Cysteine - Phenylalanine - Glycine - Glycine
Arginine - Isoleucine - Aspartic acid - Arginine -
S Isoleucine - Glycine - Alanine - Glutamine -
Serine - Glycine - Leucine - Glycine - Cysteine -
Asparagine - Serine - Phenylalanine - Arginine -
Tyrosine.
A similar methodology was employed using an Applied
Biosystems~gas phase sequencer in order to confirm the
same linear sequence. The molecular weight of this
peptide is about X0500.
It will, of course, be appreciated that the presence of
the relatively short disulfide bond between the relatively
widely spaced cysteine groups indicates that the structure
is not linear but is folded in such a manner that the
cysteine groups are sufficiently close to each other to
be bridged by the disulfide bond. It will be further
appreciated that following establishment of the amino
acid linear sequence synthesis of the peptide of the
present invention may be readily effected by standard
biochemical techniques or by recombinant DNA technology.
The ability of either crude atrial extracts or
partially purified atrial natriuretic factor to induce a
rapid and potent diuretic and natriuretic response in
bioassay animals has been noted above. In addition,
recent tissue fractionation studies, have shown that this
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134x622
activity appears largely stored in the secretory-like
specific atrial granules. These granules share histo-
chemical and tinctorial properties with granules known
to store polypeptide hormones. Histochemical data
suggest the presence of sulphur-containing amino acids
in atrial granules and cardionatrin I contains a cystine
residue and a methionine residue. However, the histo-
chemical studies also indicate the presence of indole
in atrial granules, but the amino acid composition of
cardionatrin I showed that no tryptophan was present.
The reasons for this apparent discrepancy are not clear
at present but could be related to the specificity of
the histochemical technique or the presence of other
indole containing species within atrial granules.
Clarification of this may be expected with the
characterization of the remaining peptides with
natriuretic activity found after RP-HPLC of atrial
homogenates.
The ANF of the present invention is clearly
a highly useful pharmaceutical composition for diuretic
and natriuretic purposes and is also believed to be of
great potential in reaching a better understanding of
the in vivo control of water and electrolyte balance.
Should future investigations show that peptides such as
cardionatrin I are released from the atria to modify
kidney function then this would have a major impact on
the understanding of disturbances of water and electrolyte
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13-40622
balance which occur in such clinical entities as
essential hypertension and chronic congestive heart
failure.
10
20
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