Department of Pathology, George Washington University Medical Center, Washington, District of Columbia 20037
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ABSTRACT |
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This study was concerned with the
effects of NaCl administered in vivo or added in vitro to isolated
nuclei on [3H]tryptophan binding to rat hepatic
nuclei assayed in vitro. Hypertonic (10.7%) NaCl administered in vivo
to rats caused at 10 min a marked decrease in in vitro binding (total
and specific) of [3H]tryptophan to hepatic
nuclei. In vitro incubation of isolated hepatic nuclei, but not of
isolated nuclear envelopes, with added NaCl (particularly at 0.125 × 104 M and 0.25 × 10
4 M) revealed significant inhibition of
[3H]tryptophan binding. However, isolated
hepatic nuclear envelopes prepared after in vitro incubation of
isolated nuclei with added NaCl did show inhibition of
[3H]tryptophan binding (total and specific)
compared with controls. Other salts (KCl, MgCl2,
NaHCO3, NaC2H3O2, NaF,
or Na2SO4), at similar concentrations to that
of NaCl except for MgCl2, when added to isolated nuclei did
not appreciably inhibit nuclear tryptophan binding. Kinetic studies of
in vitro nuclear [3H]tryptophan binding in the
presence of 0.125 × 10
4 M NaCl revealed that
binding decreased at 0.5 h and continued to 2 h compared with nuclear
[3H]tryptophan binding with controls (without
NaCl addition). The results obtained in vivo in rats and those obtained
in vitro with isolated hepatic nuclei revealed NaCl-induced inhibitory
effects on [3H]tryptophan binding to hepatic
nuclei. Although the inhibitory effects were similar under the two
different experimental conditions, the mechanism for each may be
different in that the NaCl concentration in hepatic cells after
administration of NaCl in vivo was appreciably higher than the low
levels added in vitro to the isolated hepatic nuclei.
added sodium chloride; nuclear envelopes; rats
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INTRODUCTION |
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THE ENHANCEMENT OF HEPATIC protein synthesis in mice, rats, rabbits, and pigs, which is rapidly induced by the administration of L-tryptophan (27, 29-33, 35), has been the central focus of our interest for many years. In attempting to unravel the mechanism involved in this stimulatory response, we have discovered that hepatic nuclei, particularly their nuclear envelopes, have a specific receptor for L-tryptophan (15, 16). This receptor binding with L-tryptophan, which is stereospecific, saturable, and of high affinity, resides predominantly in nuclear envelopes (84%), with only small levels of binding present in euchromatin (6.5%), nucleoli (3.5%), and heterochromatin (3.0%; see Ref. 15). Also, isolated nuclei, washed with buffer containing 1% Triton X-100 to remove the outer nuclear membrane, retained their binding capacity for [3H]tryptophan like that of untreated nuclei, which suggested that the inner nuclear membranes of the nuclei were vital binding sites (15). The nuclear envelope receptor binding of L-tryptophan has been considered to be of importance in the tryptophan-induced enhancement of nucleocytoplasmic transport of mRNA within liver cells, a process that has been speculated to be intimately involved in the enhanced protein synthesis (14, 17, 24, 25, 35, 36).
One important physiological process that rapidly influences hepatic protein synthesis is the tonicity of the cellular environment. Administration of hypertonic solutions to animals rapidly diminishes hepatic protein synthesis, whereas the administration of hypotonic solutions rapidly stimulated hepatic protein synthesis (10, 11, 20-22). These responses have been correlated with alterations in cell volume; hyperosmotic cell shrinkage induces stimulation of catabolic pathways, whereas hypoosmotic cell swelling enhances anabolic pathways (10, 11). Cell volume regulation is fast (generally within minutes) but leaves cells with disturbed intracellular inorganic ion concentrations that are in most cases disadvantageous. In earlier studies, we investigated the effects on hepatic protein synthesis of altered tonicity alone (20-22) or combined with the influence of L-tryptophan (38) and found that the inhibition of hepatic protein synthesis due to the administration of hypertonic salt could be negated or reversed by the administration of L-tryptophan. The latter observation suggested that the effects of L-tryptophan on protein synthesis in the livers of animals treated with hypertonic salts occurred by similar mechanisms to those involved in normal animals. However, the overall effect was somewhat less than that in normal animals (38). In this study, we investigated whether changes in tonicity within cells induced by administering in vivo increased levels of NaCl would affect or influence tryptophan binding to specific receptors in hepatic nuclei and their membranes. Also, we investigated whether added NaCl to isolated hepatic nuclei in vitro would affect their binding affinity for [3H]tryptophan. Our current findings based on in vivo and in vitro experiments indicate that the hepatic nuclear receptor affinity for L-tryptophan is highly sensitive to changes in environmental NaCl concentrations (high in vivo and low in vitro) and is the subject of this report.
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MATERIALS AND METHODS |
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Animals
Female Sprague-Dawley rats (Hilltop Laboratory Animals, Scottdale, PA) weighing an average of 175 g were used in the experiments. The rats were fed a commercial diet (Rodent Lab Chow, Purina Mills, St. Louis, MO) and were maintained in a temperature-controlled room with alternating 12:12-h light-dark cycles. Before beginning the experiments, the animals were fasted overnight but had free access to water.Experimental In Vivo Treatments
In experiments designed to investigate the effects of the administration of isotonic NaCl (0.7% NaCl), hypertonic NaCl (10.7% NaCl), hypotonic solution (distilled water), or nothing on overnight-fasted rats, animals were tube fed each of above 10 min before killing. In these experiments, rats received 3 ml solution/100 g body wt, and the hypertonic NaCl was 10.7%. Rats were killed by decapitation. These studies were approved by the institutional animal care and use committee.Chemicals
The [3H]tryptophan was L-[5-3H]tryptophan (1.13 TBq/mmol; Amersham/Searle, Arlington Heights, IL). Unlabeled L-tryptophan was obtained from US Biochemical (Cleveland, OH), and other compounds were obtained from Sigma Chemical (St. Louis, MO).Isolation of Nuclei
Rat hepatic nuclei were prepared as described by Blobel and Potter (3). The liver tissue was minced and homogenized in 2 vol of 0.05 M Tris · HCl, pH 7.5, 0.025 M KCl, 0.005 M MgCl2, 0.0001 M phenylmethysulfonyl fluoride (PMSF), 0.0002 M dithiothreitol, and 0.25 M sucrose (buffer A). The homogenate was filtered through cheesecloth (four layers) before mixing with 2 vol of 0.05 M Tris · HCl, pH 7.5, 0.025 M KCl, 0.005 M MgCl2, and 2.3 M sucrose (buffer B). The latter was underlaid with 1 vol buffer B and was centrifuged for 60 min at 105,000 g in an ultracentrifuge (model L5-75 Spinco; Beckman Instruments, Fullerton, CA). The supernatant was discarded, and the pellet was washed two times with buffer A before further use.Preparation of Nuclear Envelopes
Nuclear envelopes were isolated according to the method of Agutter and Gleed (1), which is a modified technique of that described by Harris and Milne (9) and is routinely used in this laboratory (15, 16). The purified nuclear envelopes were resuspended in a binding assay buffer containing 0.05 M Tris · HCl, pH 7.5, 0.002 M EDTA, 10% vol/vol glycerol, 0.001 M PMSF, and 0.002 MBinding of [3H]tryptophan to Nuclei or Nuclear Envelopes
Rat hepatic nuclei or nuclear envelopes prepared as described above were incubated with L-[5-3H]tryptophan (277.5 kBq, 0.245 nmol L-tryptophan/assay, added to incubation mixture last at time 0) in the absence or presence of a 2,000-fold excess of unlabeled L-tryptophan (10Other Determinations
The protein content was determined as described by Lowry et al. (19). Data were analyzed by Student's t-test (39). ![]() |
RESULTS |
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In Vivo Effects of Tonicity Changes by Varying NaCl Concentrations on Nuclear Receptor Binding of L-Tryptophan
In the first series of experiments, the effects of tube feeding isotonic (0.7% NaCl), hypertonic (10.7% NaCl), or hypotonic (distilled water) solutions or of feeding nothing to overnight-fasted rats were investigated. [3H]tryptophan binding to isolated hepatic nuclei, without or with added unlabeled L-tryptophan (10
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In Vitro Effects of Adding NaCl on Nuclear Receptor Binding of L-Tryptophan
In the second series of experiments, we investigated in vitro the effects of varying the concentrations of NaCl in the media on [3H]tryptophan binding to isolated hepatic nuclei. In these in vitro experiments, isolated nuclei were suspended in buffer A as described in MATERIALS AND METHODS and as in earlier binding studies (15, 16, 37). In early experiments, we investigated the effects of adding varying NaCl concentrations from 10Whether small changes in concentration of added NaCl (between
104 and 10
6 M) would influence in
vitro [3H]tryptophan binding to hepatic nuclei
was subsequently investigated (Fig. 1).
Specifically, the effects on [3H]tryptophan
binding due to NaCl or to unlabeled L-tryptophan at
concentrations 0.25 × 10
5 to
10
4 M were determined. In some experiments, each of
the two compounds was simultaneously tested. The findings indicate that
appreciable inhibition of binding occurred mainly between 0.375 × 10
4 to 0.25 × 10
5 M NaCl
(29.0-46.8%), with the greatest (significant) inhibition at 0.25 × 10
4 and 0.125 × 10
4
M NaCl (46.8 or 46.5%, respectively). Unlabeled
L-tryptophan at similar concentrations
(10
6 to 10
4 M) to those used with
NaCl revealed greater degrees of inhibition (from 67.4 to 42.6%
inhibition with decreasing concentrations), similar to results reported
earlier (15).
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Next, we investigated whether the in vitro
[3H]tryptophan binding changes due to the added
concentrations of NaCl reported above with isolated hepatic nuclei
would occur when using isolated hepatic nuclear envelopes. In these in
vitro experiments, isolated nuclear envelopes suspended in buffer
C were investigated as described in MATERIALS AND
METHODS and as used in earlier binding studies (15, 16). In four
experiments, hepatic nuclear envelopes were incubated with varying
concentrations of NaCl (0.25 × 105, 0.5 × 10
5, 10
5, 0.125 × 10
4, 0.375 × 10
4, 0.5 × 10
4, or 10
4 M), and in
vitro [3H]tryptophan binding was assayed. The
results revealed minimal binding inhibition (<5%) due to the
different concentrations of added NaCl. In two experiments, hepatic
nuclear envelope binding of [3H]tryptophan was
determined with varying additions of NaCl using buffer C to
which was added KCl (0.025 M) and MgCl2 (0.005 M), similar
to that used with hepatic nuclei in buffer A. The results were
similar to those described above for experiments using buffer C
and rule out the possible influence of KCl and MgCl2 salts
used in the buffer of the binding experiments with hepatic nuclei. This
indicated that the inhibitory effect on
[3H]tryptophan binding of the NaCl
concentrations that affected intact isolated nuclei did not occur when
using isolated nuclear envelopes. Earlier, we reported that the nuclear
envelopes contained the predominant level (84%) of the nuclear
tryptophan receptor (15, 16).
Next, we investigated whether hepatic nuclear envelopes obtained from
isolated hepatic nuclei that had been incubated in buffer A
with added concentrations of NaCl would be affected in subsequent assay
of [3H]tryptophan binding to the nuclear
envelopes in buffer C. In two experiments, isolated hepatic
nuclei were incubated in buffer A for 30 min with the following
additions: water; 104 M NaCl; 0.25 × 10
4 M NaCl; 0.125 × 10
4 M
NaCl, or 10
4 M unlabeled L-tryptophan.
Next, the nuclei of each group were spun down, washed, and treated to
isolate nuclear envelopes. The isolated nuclear envelopes of each group
were then incubated in buffer C with
[3H]tryptophan without or with unlabeled
L-tryptophan (10
4 M) for 2 h. The total
binding of each group was compared with that of the water control
group, which was set at 100%. Specific binding within each group was
derived from total binding minus nonspecific binding [determined
in the presence of excess unlabeled L-tryptophan
(10
4 M) relative to total binding (expressed as
%)]. The results (means of 2 experiments) revealed the following
for percentage of total and specific binding, respectively, for each
group: water, 100 and 74.5%; 10
4 M NaCl, 69.9 and
8.1%; 0.25 × 10
4 M NaCl, 73.2 and
9.3%; and 10
4 M L-tryptophan, 43.9 and
73.2%. Thus the results of the experiments where the isolated hepatic
nuclear envelopes were isolated from nuclei that had been incubated
with varying concentrations of NaCl revealed that the in vitro
[3H]tryptophan binding to the nuclear envelopes
was only moderately decreased in total binding but was markedly
decreased in specific binding.
In in vitro experiments, the effects of adding varying concentrations
of NaCl together with constant (104 M) or varying
concentrations of unlabeled L-tryptophan on
[3H]tryptophan binding to hepatic nuclei were
investigated. The addition of NaCl at varying concentrations
(10
4 to 10
10 M, but not
10
12 M) appeared to diminish the percentage of
binding inhibition due to that of unlabeled L-tryptophan
(10
4 M) alone (Table
2). In another group of
experiments, the addition of NaCl (0.5 × 10
4, 0.25 × 10
4,
10
5, 0.5 × 10
5,
or 0.25 × 10
5 M) to comparable concentrations
of unlabeled L-tryptophan caused significant decreases in
binding inhibition compared with that of the unlabeled
L-tryptophan alone at all of the concentrations except at
the 0.25 × 10
5 M level (Table 2). In one
experiment, we used unlabeled L-tryptophan at
10
4 M with limited varying levels of NaCl
(10
4, 0.5 × 10
4, 0.375 × 10
4, 0.25 × 10
4,
0.125 × 10
4, 10
5, 0.5 × 10
5, or 10
6 M), and the
results revealed inhibitions of 38.2-45.8% for all tests
concentrations, indicating no major changes with the different concentrations of NaCl.
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Next, we investigated whether sodium salts other than NaCl or other
salts with or without chloride would act similarly or differently than
that of equimolar concentrations of NaCl on in vitro
[3H]tryptophan binding to hepatic nuclei. In
comparison with the inhibitory binding effects of the addition of NaCl
at 104 to 0.25 × 10
5 M
described in Fig. 1, the effects on binding inhibition of KCl, NaHCO3, NaC2H3O2, NaF,
or Na2SO4 were only minimal at the
concentrations studied, except for MgCl2, which caused a
24% inhibitory effect at 0.5 × 10
4 or 0.375 × 10
4 M (0.05 > P > 0.02) and a
21% inhibitory effect at 0.25 × 10
5 M
(P < 0.01; Table 3). It is
important to note that the usual incubation medium (buffer A)
in all of the incubation [3H]tryptophan binding
experiments using hepatic nuclei contained 0.025 M KCl and 0.005 M
MgCl2, and the tested concentrations of KCl and
MgCl2 were therefore the sums of that added to that which was already present in the incubation medium. When adding
10
4 M KCl, the total concentration became 251 × 10
4 M KCl, and when adding
10
4 M MgCl2 the total concentration
became 51 × 10
4 M. Our experiments indicate
that NaCl has a specific inhibitory effect on binding (Table 2) that
cannot be attributed to either the sodium ions alone or to chloride
ions alone.
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Further Studies with Adding NaCl to Isolated Hepatic Nuclei
In attempting to obtain insight into how the in vitro addition of NaCl acts to inhibit [3H]tryptophan binding to isolated hepatic nuclei, further experiments were concerned with the saturation isotherm, with the kinetics of tryptophan binding and release, with tryptophan levels, and with the effect of addition of L-leucine.Saturation isotherm.
The saturation isotherm for [3H]tryptophan
binding using unlabeled L-tryptophan
(104 M) without or with added NaCl (0.125 × 10
4 M) is presented in Fig.
2. It is apparent that, at each of the concentrations of [3H]tryptophan used, the
added NaCl (0.125 × 10
4 M) to the unlabeled
L-tryptophan (10
4 M) diminished the
specific binding to hepatic nuclei (24-52%).
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Kinetics of regular binding.
In two experiments, hepatic nuclei were incubated with
[3H]tryptophan in the presence of the addition
of water, unlabeled L-tryptophan (104
M), or NaCl (0.125 × 10
4 M) for intervals of
0.5, 1, 1.5, or 2 h. The findings expressed as percentage binding at
each interval indicate that there was diminished
[3H]tryptophan binding to hepatic nuclei by
unlabeled L-tryptophan or NaCl at each of the four
intervals (Fig. 3). However, there was a
greater percentage inhibition due to unlabeled L-tryptophan (67-72%) than that due to NaCl (50-56%) at each interval.
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Kinetics of [3H]tryptophan binding after
preincubation with NaCl.
In one experiment, we investigated the effects of preincubation of
hepatic nuclei with added water, unlabeled L-tryptophan (104 M), or NaCl (0.125 × 10
4 M) for 5, 15, 30, or 60 min on subsequent
[3H]tryptophan binding to hepatic nuclei for 2 h. In this experiment, after the initial incubation of nuclei of each
group in media without [3H]tryptophan for the
times indicated, nuclei were spun down, washed, and then resuspended in
media containing [3H]tryptophan and were
incubated for 2 h. The results indicated that preincubation caused a
decrease in the percentage binding, beginning at 5 min and bottoming
out at 30 min. In all cases, the inhibition of binding was greater with
unlabeled L-tryptophan than with NaCl. For each time
interval, compared with the water group after 5 min (set at 100%), the
results for water, unlabeled L-tryptophan, and NaCl,
respectively, at each time interval were as follows: 5 min, 100, 27.7, and 50.7%; 15 min, 70, 26.7, and 39.6%; 30 min, 52.7, 14.7, and
28.5%; and 60 min, 65, 17.8, and 35.7%.
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Kinetics of release of [3H]tryptophan
binding.
In several experiments, the effect of added NaCl (0.125 × 104 M) on nuclear
[3H]tryptophan binding (total and nonspecific)
release was investigated. Here hepatic nuclei with or without added
unlabeled L-tryptophan (10
4 M) were
incubated with [3H]tryptophan for 2 h. Nuclei
of each group were then spun down, washed with buffer, and spun down
again. The control groups were then counted. Other groups of hepatic
nuclei were then incubated in buffer A to which was added NaCl
(0.125 × 10
4 M) or water for 15 min. Nuclei
were then spun down, washed, spun down, and counted. The results are
summarized in Table 5. Loss of bound
[3H]tryptophan to nuclei (total and
nonspecific) was essentially unchanged during the 15-min additional
incubation of the water (control) group. However, 15 min of incubation
with NaCl (0.125 × 10
4 M) induced a
significant loss of total bound counts, with an increase in nonspecific
bound counts. This indicated that the addition of NaCl induced an
appreciable decrease in specific bound counts.
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Free tryptophan levels in hepatic nuclei.
To determine whether incubation with varying levels of NaCl
(105 to 10
4 M) added to the in
vitro assay of [3H]tryptophan binding to
hepatic nuclei may affect the free tryptophan levels in the binding
media, we measured the free tryptophan levels after a 2-h binding
incubation. After incubation, the nuclei were spun down, and the
supernatants were then assayed for free tryptophan levels. In one
experiment, the tryptophan levels in controls (water group) were
compared with those incubated with added NaCl at
10
4, 0.5 × 10
4, 0.25 × 10
4, 0.125 × 10
4, or 10
5 M. Essentially no
differences in free tryptophan levels were observed among the groups.
Effect of the addition of L-leucine.
In an earlier study (37), we reported that, although
L-leucine did not compete for in vitro
[3H]tryptophan binding to hepatic nuclei, the
addition of L-leucine to excess unlabeled
L-tryptophan (104 M) prevented the
inhibition of [3H]tryptophan binding that
occurred due to the unlabeled L-tryptophan alone.
Therefore, in this study, we investigated whether the addition of
L-leucine (10
4 M) would affect the in
vitro [3H]tryptophan binding to hepatic nuclei
as influenced by the addition of NaCl (0.25 × 10
4 or 0.125 × 10
4 M). The
results of such experiments were as follows [expressed as
%inhibition of binding compared with control (water) group set at
100% with number of experiments in parentheses]: 1) 0.25 × 10
4 M NaCl alone, 44.0 ± 2.4% (n = 5); 2) 0.25 × 10
4 M NaCl
plus 10
4 M L-leucine, 16.0 ± 1.1%
(n = 4); 3) 0.125 × 10
4 M NaCl
alone, 47.8 ± 2.2% (n = 4); 4) 0.125 × 10
4 M NaCl plus 10
4M
L-leucine, 12.7 ± 3.2% (n = 5); and 5)
10
4 M L-leucine alone, 8.3 ± 4.1%
(n = 5). Thus it is apparent that the addition of
L-leucine negated the inhibitory binding effects of the
addition of NaCl (0.25 × 10
4 or 0.125 × 10
4 M).
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DISCUSSION |
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Our in vivo experiments revealed that the administration by tube feeding a hypertonic solution (10.7% NaCl) rapidly (within 10 min) caused a significant decrease in the ability of hepatic nuclei to bind [3H]tryptophan (total and specific) compared with controls when assayed by in vitro incubation (Table 1). This in vivo effect can most probably be attributed to the rapid increase in NaCl concentration in the vascular spaces and within hepatocytes of the liver. In earlier studies where mice were tube fed a hypertonic solution (4% NaCl) 10 min before death, the portal blood plasma osmolality increased (23%), the liver weight decreased (9%), and liver sodium content increased (16%; see Ref. 22). Also, Bedford and Leader (2) reported that in rats the intraperitoneal infusion of hypertonic NaCl (1 M or 5.8%) for 1 h caused elevations in sodium content (+7%) and in total osmolarity [mosmol/kg dry wt (+70)]. Thus the altered extracellular, cellular, and cytosolic NaCl concentrations invariably may affect the hepatic nuclei such that their affinity to bind [3H]tryptophan as assayed in vitro becomes diminished. The literature contains many examples whereby changes in tonicity affect membrane binding to liver and other organs or cells (5, 6, 8, 12, 13, 23, 44).
Earlier, we reported that rats tube fed a hypertonic solution (6.2 or 10.7% NaCl) 10 min before death revealed decreased polyribosomal aggregation and decreased protein synthesis in the liver (38). On the other hand, simultaneous treatment with L-tryptophan and hypertonic NaCl for 30 min improved polyribosomal aggregation and protein synthesis of the liver compared with that in rats receiving hypertonic NaCl alone (38). This improvement was less than the effect of L-tryptophan alone on control (no NaCl treatment) rats. Other experiments revealed that the administration of L-tryptophan before (30 min) or after (30 min) the administration of hypertonic solution (6% NaCl) improved hepatic polyribosomal aggregation and protein synthesis compared with that in animals receiving hypertonic NaCl alone (34). In our present in vivo experiments, tube feeding of hypertonic NaCl (10.7%) rapidly (within 10 min) decreased the affinity for in vitro [3H]tryptophan binding to hepatic nuclei (total and specific binding), yet some hepatic nuclear receptors for tryptophan remained (Table 1). Thus the in vivo responses of rat livers to L-tryptophan alone reported earlier (30), as well as those of rat livers exposed to hypertonic NaCl and L-tryptophan, may be explained by the effects of L-tryptophan as occurs in normal animals. Support for this comes from earlier findings that mRNA (38) and in vitro nuclear RNA efflux (34) in the livers of hypertonic NaCl-treated rats were increased due to the administration of L-tryptophan as had been reported earlier in normal rats (24, 25, 36).
In the present study, we also explored whether in vitro additions of
NaCl to isolated hepatic nuclei or nuclear envelopes would affect their
[3H]tryptophan binding. Our findings indicated
that the addition of certain low concentrations (0.125 × 104 or 0.375 × 10
4 M) of
NaCl affected isolated hepatic nuclei in their binding affinity for
L-tryptophan as well as morphologically. The inhibitory binding effect was on hepatic nuclei but did not occur with NaCl added
to isolated hepatic nuclear envelopes as determined experimentally. However, hepatic nuclear envelopes isolated after treatment of hepatic
nuclei with added NaCl did demonstrate decreased binding affinity as
occurred when using intact hepatic nuclei. Thus the findings are of
special interest in that the hepatic nuclear receptor protein for
L-tryptophan has been reported to be present
predominantly in the isolated nuclear envelopes of livers of normal
animals (16).
In two preliminary experiments (unpublished observations), we conducted
morphological (light and transmission electron microscopy) studies on
isolated hepatic nuclei that were incubated in the presence of either
added water, tryptophan (104 M), or NaCl (0.5 × 10
4, 0.375 × 10
4,
0.25 × 10
4, 0.125 × 10
4, or 10
5 M). Nuclei incubated
with added NaCl revealed marked variations in the chromatin appearance
compared with controls, but the variations were such that it was
difficult to quantitate the alterations among the nuclei of the
different groups (like to concentrations of added NaCl). Overall, the
preliminary findings suggested condensation of the chromatin due to
added NaCl. Such alterations in nuclear chromatin could disturb the
normal relationship between the inner nuclear envelope and its internal
components (chromatin) and thereby could affect the nuclear envelope
binding affinity for tryptophan. The nuclear lamina, a filamentous
protein meshwork lining the inner nuclear membrane, is considered to
provide a structural framework for the nuclear envelope and an
anchoring site for chromatin at the nuclear periphery (7). Yang et al.
(45) have described a lamin-associated polypeptide 2, which is an
integral membrane protein of the inner nuclear membrane that binds to
both lamin B and chromatin and which controls nuclear volume increases.
Small additions of NaCl may disturb this relationship.
Salt-induced modifications of rat liver nuclear chromatin have been described by raising NaCl concentrations (40). However, such changes usually develop at high NaCl concentrations as with 600 mM (40). In our present study, the additions of NaCl in vitro to isolated intact hepatic nuclei were at much lower levels. However, these NaCl additions in an incubation medium (buffer A) that contained other ions and salts (KCl and MgCl2) could trigger configurational changes in the chromatin. It is known that condensation of chromatin depends on the ion composition in the cell nucleus. Oberleithner et al. (26) tested the influence of various ions on nuclear volume and intranuclear voltage of isolated nuclei of Madin-Darby canine kidney cells. They stressed that changes in the composition and functional state of nuclear chromatin were of great importance in the response found due to their experimentally altered parameters.
Of vital importance in interpreting our results is whether the concentrations of NaCl used in our in vitro studies with isolated hepatic nuclei were within the physiological range that exists within living intact hepatic cells. Review of earlier reports by others reveal that, in normal rat liver, the concentration of sodium ranges from 35 to 87 mmol/kg liver or on the average 61 mM (2, 41). Thus this level probably reflects the sodium concentration in the cytosol of liver cells as well as of blood in hepatic vessels and sinusoids. Our own earlier determination of liver sodium concentration as assayed on levels in postmitochondrial supernatants of control rats was 12.9 mM (22). The cited values do not address what the true physiological levels of sodium are in hepatic nuclei. Our in vitro incubation medium contained five added components at a concentration of 330 mM. To this was added 12.5-100 µM NaCl. Overall, such sodium levels are extremely low compared with the levels in liver as a whole. However, based upon our current in vitro studies with isolated hepatic nuclei, it appears that additions of low levels of NaCl to our incubation medium can affect the binding affinity for L-tryptophan as well as the structural integrity of the nuclei. Further studies dealing with NaCl effects on isolated hepatic nuclei are warranted and may in future studies clarify why and how such low concentrations of NaCl (compared with that normally in hepatic cytosol) can be of importance on hepatic nuclear receptor binding of L-tryptophan in an artificial in vitro environment.
Although we have observed similar effects in the altered binding affinity of L-tryptophan to hepatic nuclei after treatment with NaCl of intact animals and of isolated nuclei, the mechanism(s) involved is/are most probably different under the two conditions. In the first case, rats were tube fed a hypertonic NaCl solution that rapidly induced vascular, intercellular, and intracellular tonicity changes in the liver. In the second case, isolated hepatic nuclei were exposed to low concentrations of NaCl. Behavior of isolated nuclei may be quite different in an artificial media from that within intact cells. Indeed, Robbins et al. (28) described morphological alterations (chromosome condensation with preferential localization at the nuclear envelope and nucleolus) in interphase HeLa cells exposed to hypertonic NaCl (1.6× isotonic). However, such changes were not observed by them when isolated nuclei were exposed to a hypertonic NaCl medium. They concluded that the nuclear response in the intact cell may be partly dependent on unknown molecules inadvertently lost to the medium during separation of nuclei from cytoplasm. Others have also described morphological transformation in V79 Chinese hamster cells after hypertonic NaCl treatment (43), which included ruffling and indentation of the nuclear membrane, structural modifications of the nucleolus, and chromatin condensation in a prophase-like pattern [as defined by Robbins et al. (28)]. Also, Laval and Bouteille (18) reported that, throughout their ultrastructural study of rat liver nuclei during isolations and then during incubation in vitro, remarkable morphological changes appeared to be related to the ionic concentration; the chromatin is alternately condensed and dispersed, and, finally, in the incubation medium, the internal structure shows severe clumping that is reversible.
In recent years, cell volume changes have been recognized as potent modulators of metabolic liver cell function (42). Such changes clearly have been implicated in altering hepatic protein synthesis (10, 11). Hypertonicity causes an osmotic efflux of water that shrinks cells and rapidly diminishes protein synthesis (10, 11). Also, cells react to increased osmolality with numerous changes in gene expression (4). Thus regulation of osmolarity has been determined to be one of many important cellular metabolic controls. Therefore, in the present study, we have investigated whether the effects of cellular hyperosmolarity due to the administration of NaCl, particularly relating to nuclei of the liver, may influence another regulatory control system pertaining to L-tryptophan, specifically to its ability to bind to a specific receptor present in the nuclear membrane (15, 16). Our findings indicated that the administration of hypertonic NaCl in vivo has an inhibitory effect on the binding affinity of L-tryptophan to hepatic nuclei. However, even though the administration of hypertonic NaCl inhibits protein synthesis and diminishes nuclear receptor binding of L-tryptophan, the administration of L-tryptophan to animals can overcome, or partially negate, the NaCl-induced inhibitory effect on hepatic protein synthesis (34, 38). Thus the regulatory effects of important components, such as NaCl either by inducing tonicity changes or by acting itself on nuclei and as L-tryptophan in regard to affecting nuclear efflux of mRNA (24, 35, 36) can come into play and influence the overall effect of each compound independently. Our present findings stress the complexity and interrelationships of actions of regulatory systems within cells.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Sidransky, Dept. of Pathology, George Washington Univ. Medical Center, 2300 Eye St., N.W., Washington, DC 20037.
Received 3 June 1999; accepted in final form 4 January 2000.
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