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Effects of salt loading on the fractional volume of atria-specific granules in dahl salt-sensitive and salt-resistant rats.

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THE ANATOMICAL RECORD 218:157-161(1987)
Effects of Salt Loading on the Fractional Volume of
Atria-Specific Granules in Dahl Salt-Sensitive and
Salt-Resistant Rats
JOHN T. HANSEN, J.R. HAYWOOD, AND ANDREW HOWELL
Department of Neurobiology and Anatomy, University of Rochester School of Medicine and
Dentistry, Rochester, NY 14642 (2.
TH., A.H.); Department of Pharmacology, The University
of Texas Health Science Center, S a n Antonio, T X 78284 (J.R.H.)
ABSTRACT
The cardiac atria are known to play a role in blood volume homeostasis, secreting a peptide that induces a potent natriuresis and diuresis. This
peptide is atrial natriuretic factor (ANF), and its primary site of storage is within
atria-specific granules found in atrial cardiocytes.
Since salt loading results in a n increase in circulating levels of ANF, our aim was
to determine if the atria-specific granule population in the cardiocytes of Dahl rats
would decrease accordingly. To this end, the fractional volume of the atria-specific
granules was determined by ultrastructural morphometric analysis in the Dahl salt
model of hypertension. This analysis was performed on the right atria of Dahl saltresistant (DR) and salt-sensitive (DS) rats fed either a low-salt (0.4%) or high-salt
(8%)diet for 12 weeks prior to sacrifice. DR and DS rats fed a low-salt diet had
significantly reduced plasma sodium levels and osmolalities, and a significantly
lower mean arterial blood pressure than did rats fed a high-salt diet. The fractional
volume of atria-specific granules was significantly lower in salt-loaded DR (P< 0.01)
and DS (P<O.O25) rats than in their respective low-salt controls. This significant
decrease in atrial granules corresponds to the reported decrease in the storage of
atrial ANF in salt-loaded rats, and provides a morphological verification of the
biochemical studies. Moreover, these results, in combination with a growing body of
physiological data, lend support to the hypothesized role of ANF in the regulation of
water-electrolyte balance, which may play a n important role in cardiovascular
pathophysiological states related to hypertension.
Mammalian atrial cardiocytes contain secretory granules characteristic of endocrine cells. These atria-specific
granules were first discovered by Kisch (1956) in guinea
pig heart, and later in other mammalian species (Bompiani et al., 1959; Jamieson and Palade, 1964; Hibbs and
Ferrans, 19691, including humans (Battig and Low,
1961). The granules possess a homogenous, electrondense core, are surrounded by a limiting membrane, and
measure 250-500 nm in diameter (Jamieson and Palade, 1964; Hibbs and Ferrans, 1969). Often, the granules are more concentrated in the central sarcoplasmic
core, associated with a n extensive Golgi complex located
at each nuclear pole. Rough endoplasmic reticulum, glycogen, and numerous mitochondria commonly are associated with the granule accumulations.
Atria-specific granules incorporate 3H-leucine (Yunge
et al., 1980) with kinetics similar to that found in endocrine cells that produce polypeptide hormones. Moreover, the cardiac atria are known to play a role in blood
volume homeostasis, and the number of atria-specific
granules is altered by experimental paradigms that affect water and electrolyte balance (Marie et al., 1976; de
Bold, 1979). On the basis of these and other histochemical studies (see de Bold, 1985,1986), it has been hypoth0 1987 ALAN R. LISS, INC
esized that the atria-specific granules contain basic
polypeptides involved in water-electrolyte balance, and
that mammalian atrial cardiocytes synthesize, store, and
release these substances in a manner common to other
polypeptide hormone-producing cells (de Bold, 1986).Recent immunocytochemical (Cantin et al., 1984; Chapeau
et al., 1985; Maldonado et al., 1986) and biochemical
studies have established that the atria-specific granules
store a group of peptides with molecular weights ranging from about 2,500 to 13,000 that are commonly referred to a s atrial natriuretic factor (ANF) (Seidah et al.,
1984; de Bold, 1986). Both large-molecule and smallmolecule forms of atrial natriuretic peptides exist, but
the most abundant species in atrial extracts is a 126amino acid peptide called cardionatrin IV,or y-atrial
natriuretic peptide (de Bold, 1986). The most common
low-molecular-weight natriuretic peptide isolated from
the rat atria is the 28-amino acid peptide called cardion-
Received October 14, 1986; accepted January 12, 1987.
Address correspondence to: John T. Hansen, Ph.D., Department of
Neurobiology and Anatomy, Box 603, University of Rochester School
of Medicine, 601 Elmwood Avenue, Rochester, NY 14642.
158
J.T. HANSEN, J.R. HAYWOOD, AND A. HOWELL
159
ATRIAL SPECIFIC GRANULES IN DAHL RATS
TABLE 1. Comparative data from Dahl rats fed low- or high-salt diets
Group
Mean arterial pressure Plasma Na Plasma K Osmolality
(mm Hg)
(mEqlliter) (mEq/liter) (mOsm)
DR-low
DR-high
DS-low
____
DS-high
112.6 k 3.3
124.9 f 2.01
137.6 f 1.7
179.0 f 13.5l
135.0 f 1.8
141.9 f 0.8’
137.0 f 1.6
144.6 f 0.8’
6.0
4.8
6.0
4.6
f 0.3
f 0.l2
& 0.2
f 0.l2
276
290
285
294
k2
k l2
&3
k Z3
Fractional volume
ANF granules
(%I
1.74 k 0.17
1.08 f 0.17’
1.78 f 0.18
1.18 f 0.14’
All values are means f SEM (N = 4 animals per group).
‘P <: 0.025 compared to its respective low-salt group.
‘P < 0.01 compared to its respective low-salt group.
3P i0.05 compared to its respective low-salt group.
atrin I (Flynn et al., 1983).Extracts of mammalian atria
containing ANF induce a potent natriuresis and diuresis in the intact rat (de Bold et al., 1981), which is
accompanied by hypotension, often bradycardia, and a
reduction in aldosterone and renin secretion. Additionally, ANF may be a vasoactive substance that counteracts the effects of endogenous vasoconstrictors, such as
norepinephrine and angiotensin 11, thereby leading to
the observed natriuresis (Kleinert et al., 1984).
Previous studies on ANF have been directed at elucidating the structure and normal physiological properties of this hormone. Recently, attention has been
redirected toward understanding the role of ANF in
cardiovascular pathophysiological states related to hypertension. Of special interest is the relationship of ANF
to volume-overload hypertension. Hirata and co-workers
(1984) have investigated the role of ANF in the Dahl
salt model of hypertension, where the hypertension is
hypothesized to be due in part to a humoral factor that
affects sodium excretion, blood pressure, and vascular
reactivity. The present study was undertaken to determine if volume-overload hypertension in the Dahl salt
model affected the atrial cardiocyte granule population,
a s it is reported to do in other experimental paradigms
involving water-electrolyte balance (Marie et al., 1976;
de Bold, 1979).
divided into two groups of 4 rats each and fed a diet low
in salt (0.4% NaC1; Teklad, Madison, WI) (this group
referred to hereafter as DR-L) or a high-salt diet (8%
NaC1; Teklad) (group DR-H). The DS rats likewise were
divided into two groups of 4 rats each and fed the identical low-salt (DS-L)and high-salt diet (DS-H).All groups
were fed their respective diets for 12 weeks; the animals
on a low-salt diet received distilled water ad lib, and
those on a high-salt diet received tapwater ad lib. At the
end of the 12-week period, the rats were anesthetized
with methoxyflurane gaseous anesthesia and prepared
with femoral arterial and venous catheters. A venous
blood sample was taken for plasma osmolality determinations using freezing-point depression, and sodium and
potassium determinations were made by flame photometry. In addition, the mean arterial pressures were measured directly in conscious animals via the femoral
artery catheter.
Electron Microscopy
Following the physiologicaI measurements, the rats
were anesthetized and sacrificed by intracardiac perfusion of a fixative solution containing 3% glutaraldehyde,
1%paraformaldehyde in 0.1 M sodium phosphate buffer
(pH 7.2, room temperature). The fixative was preceded
by a saline flush. Following a 10-min perfusion, the
heart was removed and immediately immersed in fresh
MATERIALS AND METHODS
fixative for a n additional 4-6 h r a t 4°C. The heart then
Male Dahl salt-resistant (DR) rats and salt-sensitive was washed in 0.1 M phosphate buffer containing 5%
(DS) rats were obtained from the Brookhaven National sucrose (pH 7.2). Prior to postfixation, the auricular porLaboratories, Upton, New York. The DR animals were tion of the right atrium was removed from each heart,
and sectioned with a razor blade into small pieces (2 mm
x 2 mm). Then the samples were postfixed in 1%osmium tetroxide, 1.5% potassium ferrocyanide in buffer
at 4°C for 16 hr. Following a maleate buffer wash, the
tissues were stained en bloc in 0.5% uranyl acetate in
0.1 M maleate buffer (pH 6) for 3 hr. The samples then
Fig. 1. Electron micrograph of atrial cardiocyte showing atria-spewere
rinsed in maleate buffer, dehydrated in a graded
cific granules (ag) distributed preferentially in a juxtanuclear location.
Golgi complexes and mitochondria (m) are common features in these series of acetones, infiltrated, and embedded in Spurr’s
cardiocytes. This cardiocyte is sectioned longitudinally and clearly resin.
demonstrates the preferential distribution of granules near the center
of the cell. x 15,000.
Fig. 2. Electron micrograph that shows the accumulation of atriaspecific granules (ag) ad,jacent to a Golgi complex. Note the variable
size and density of the granules. m, mitochondrion. ~ 2 6 , 2 5 0 .
Fig. 3. Electron micrograph showing granule profiles that appear to
be “budding” off (arrows) from the cisternae of a Golgi complex. m,
mitochondrion. ~76,000.
Morphometric Analysis
Sections 1 pm thick were cut with glass knives and
stained with toluidine blue, and sections from blocks
with atrial cardiocytes in a cross-sectional orientation
were selected for thin sectioning. Thin sections of crosssectional cardiocytes were chosen because of the anisotropic nature of atrial granule distribution when viewed
160
J.T.HANSEN, J.R. HAYWOOD, AND A. HOWELL
in longitudinal sections (see Fig. 11, i.e., atria-specific
granules resided primarily in a juxtanuclear position.
While sampling longitudinally oriented cardiocytes that
contain a nucleus would increase the granule counts,
such a n analysis would not be random. since only atrial
granules in the approximate “center” of the cardiocyte
would be sampled (Cantin et al., 1979). Cross-sectional
analysis, on the other hand, randomly samples all portions of the cardiocyte without preferentially focusing
on the center of the cell.
For each rat in each of the four groups, 5-10 electron
micrographs were collected at a n initial magnification
of ~ 4 , 0 0 0 The
.
micrographs were collected by systematic random sampling (Weibel, 1979)from the first tissue
encountered over a n open grid square. The electron microscope was calibrated with a ruled diffi-action grating
containing 2,160 lines per 1 mm. Each electron micrograph was photographically enlarged on 8-in. x 10-in.
,
yielded a
paper to a magnification of ~ 1 4 , 0 0 0which
total area of 234 pm of tissue per print. The fractional
volume of atria-specific granules in the cardiocyte was
determined by point counting, using a transparent overlay containing 837 points. All prints were coded so
that the observer collecting the data was unaware of
group identity. Repeat counts demonstrated a reliability
of about 2%, which was deemed acceptable. The fractional volume occupied by the granules was expressed
as the mean of all prints from each group (N = 20-30)
plus or minus the standard error of the mean. Comparisons of all groups were performed using a two-way analysis of variance in a 2 x 2 factorial design, with strain
(resistant or sensitive) and diet (low- or high-salt) as
independent variables. Statistical comparisons of the
DR-L and DR-H, and the DS-L and DS-H groups (diet a s
the only variable) were performed by means of a Student’s t test. A probability of P<O.O5 was considered
significant.
RESULTS
The mean arterial blood pressure, plasma sodium and
potassium levels, and plasma osmolality of all groups of
animals just prior to sacrifice are shown in Table 1.All
values were significantly different between DR-L and
DR-H groups, and between DS-L and DS-H groups. The
mean arterial blood pressure between the DR-L and DSL groups was also significant (P< 0.002). Additionally,
the mean arterial blood pressure was considerably elevated in DS-H rats (range: 143-218 mm Hg).
Estimates of the fractional volume of the atria-specific
granules in the cardiocytes is shown in Table 1. The
fractional volume of granules was significantly elevated
(P< 0.01) in DR-L rats compared to the DR-H group. The
DS-L rats also exhibited a significant increase (P< 0.025)
in cardiocyte granules compared to the DS-H group.
Two-way ANOVA showed a significant effect of diet, but
no significant effect of strain or straiddiet interaction
on the fractional volume of atria-specific granules.
Atria-specific granules often were distributed preferentially in a juxtanuclear location (Fig. l),although
scattered granules also were observed throughout the
cardiocytes and along the sarcolemma. Most atria-specific granules displayed a homogeneous core that was
surrounded by a unit membrane (Figs. 1-3). The granules were especially abundant in the area around the
Golgi complex (Fig. 2), and they often varied in size and
in the density of their granule core. In some instances,
profiles were observed that resembled granules “budding” off from the cisternae of the Golgi complex (Fig.
3). With the exception of the documented estimates of
atria-specific granule distribution, no other differences
in morphology were observed among any of the groups.
DISCUSSION
Immunocytochemical and radioimmunoassay techniques have shown that ANF is stored within atriaspecific granules (Cantin et al., 1984; de Bold, 1985).
Water deprivation decreases the atrial content of messenger RNA for precursor ANF (Nakayama et al., 1984)
and increases the cardiocyte content of atria-specific
granules (de Bold, 1979). Therefore, one might hypothesize that during water deprivation a high atrial content
of ANF is the result of a decrease in demand, a n increase
in storage, and a decrease in de novo synthesis of ANF.
Conversely, the number of atria-specific granules decreases after saline treatment (de Bold, 1979). Apparently, salt loading increases the demand for ANF, which
results in a n increase in blood levels of ANF and a
decrease in the atrial content. In this instance, one might
hypothesize that salt loading reflects a state of rapid
synthesis and release of ANF, and concomitant decrease
in storage (Snajdar and Rapp, 1985).Therefore, salt loading has the opposite effects of water deprivation on ANF
synthesis, storage, and release. If one assumes that the
number of atria-specific granules in a cardiocyte is related to the content of ANF in that cardiocyte, our results in Dahl rats support these two hypotheses with
respect to the atrial storage of ANF. Previous studies
support the basic assumption that atria-specific granule
numbers and the levels of ANF in the atria are related,
suggesting that changes observed in atria-specific granule density under different experimental paradigms is a
reflection of relative ANF content (Marie et al., 1976; de
Bold, 1979). In the DR-H and DS-H rats, atria-specific
granules are significantly reduced compared to their
respective low-salt controls. Plasma sodium and potassium levels and osmolality of the salt-fed groups are
normal, but the mean arterial blood pressure is significantly elevated. The low-sodium-fed groups not only had
more atria-specific granules, but plasma sodium and
osmolality also are significantly reduced. Therefore, the
fractional volume of atria-specific granules in Dahl rats
subjected to high- and to low-salt intake follows the
expected and hypothesized pattern.
Hirata et al. (1984) have demonstrated that DS rats
have more ANF than DR rats. If atria-specific granule
concentration and ANF content can be correlated, as
suggested by others (see de Bold, 1985), it is interesting
that our DS rats showed a tendency to possess a greater
fractional volume of atria-specific granules than did DR
rats. However, these differences are slight, not significant, and probably simply represent variability within
our sample population. The analysis of variance shows
no significant interaction of strain and diet. Only diet
appears to have a significant effect on atria-specific
granule fractional volume estimates. Our inability to
demonstrate a correspondingly higher granule fractional volume in the DS strain than in the DR strain
could be the result of several factors. First, with the
ATRIAL SPECIFIC GRANULES IN DAHL RATS
electron microscope we have quantitated only the fractional volume of atria-specific granules. Nothing can be
concluded about the amount of ANF in each granule
(compare Figures 2 and 3) (Marie et al., 1976; Cantin et
al., 1979). Second, DS rats appear to need more ANF
because their kidneys have about a 50% lower natriuretic response to ANF compared to DR rats (Hirata et
al., 1984). Moreover, Snajdar and Rapp (1985) have
shown that DS rats not only have kidneys hyporesponsive to the effects of ANF but also tend to release less
ANF from their atria. Hence, DS rats usually show a n
increased amount of ANF in their atria. Apparently, our
morphometric approach is not sensitive enough to detect
this strain difference. Alternatively, diet appears to play
a significant role in the granularity of the atrial cardiocytes, and this effect may simply mask any strain differences that might otherwise appear. This possibility could
be tested by simply comparing DR and DS strains without the complicating influence of a low- or high-salt diet.
Finally, the absolute number of atria-specific granules
per atrial cardiocyte may be larger in DS rats. This
would be true if the atrial cardiocytes hypertrophied
secondary to a n increased blood pressure. Based upon
cross-sectional area, the atrial cardiocytes do not appear
to be larger in DS rats, although their longitudinal size
may be increased. In fact, the wet weights of the hearts
in the DS-L rats is 24% greater than in the DR-L animals, and the DS-H hearts weigh 57% more than the
DR-H hearts. How much of this increase in the wet
weight of the heart is attributable to a n increase in the
ventricle was not determined in this study. However, if
DS rats do possess larger atrial cardiocytes, the fact that
the relative fractional volume data are similar for both
groups on the same diet reflects that the absolute
amount of atria-specific granules may be increased in
the DS strain.
This study provides a morphological verification of the
reported decrease of atrial ANF and plasma volume
regulation in Dahl rats during high-salt intake. These
results, in combinat,ion with a growing body of physiological data, lend further support to the hypothesized
role of ANF in the regulation of sodium and water balance. Future discoveries that support such a role for
ANF may provide the basis for new therapies for hypertension and congestive heart failure (de Bold, 1985; Ballermann and Brenner, 1986; Cantin and Genest, 1986).
ACKNOWLEDGMENTS
The authors would like to thank Ms. Nancy A. Ball
for her excellent technical assistance. This research was
supported by U S . Public Health Service grants HL
36038 (J.T.H.) and HL 33937 (J.R.H.). Dr. Hansen is the
recipient of a National Institute of Health Research
Career Development Award.
161
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