Effects of altered tonicity by sodium chloride on L-tryptophan binding to hepatic nuclei

Herschel Sidransky, Ethel Verney, and Jan Orenstein

Department of Pathology, George Washington University Medical Center, Washington, District of Columbia 20037


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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 × 10-4 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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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 M beta -mercaptoethanol (buffer C).

Binding 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 (10-4 M) or test compound (10-4 M) at room temperature for 2 h. Nuclei were then washed (by resuspension and centrifugation) three times with buffer A, and nuclear envelopes were washed two times with buffer C to remove free and loosely bound radioactivity. After the final wash, nuclei or nuclear envelopes were suspended in buffer A or buffer C, respectively, and then radioactivity was measured after adding aqueous counting scintillation fluid (Opti-Fluor; Packard Instrument, Meriden, CT). Binding of [3H]tryptophan to hepatic nuclei or nuclear envelopes was expressed as counts per minute per unit protein. Values were compared with values obtained using unlabeled L-tryptophan (control group). In some experiments, varying concentrations (10-12 to 10-4 M) of test compounds were used.

Other Determinations

The protein content was determined as described by Lowry et al. (19). Data were analyzed by Student's t-test (39).


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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 (10-4 M), from rats killed 10 min after treatment was assayed. All rats that were tube fed received 3 ml solution/100 g body wt. The changes in in vitro [3H]tryptophan total and specific binding to hepatic nuclei of the groups that were tube fed the solutions compared with that in the untreated group were as follows: due to 0.7% NaCl, -3.2 and -19.6%; due to hypertonic NaCl, -41 and -63.8%; and due to distilled water, -20.2 and -32.3% (Table 1). The specific binding was determined from total binding minus nonspecific binding [binding in the presence of excess (10-4 M) unlabeled L-tryptophan]. The changes due to the administration of hypertonic NaCl solution or distilled water occurred rapidly, within 10 min. In an earlier study (20), we reported that, within 10 min after the administration of hypertonic NaCl solutions, the portal blood tonicity and liver sodium levels revealed significant elevations.

                              
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Table 1.   In vitro [3H]tryptophan binding to hepatic nuclei of rats tube fed nothing, 0.7% NaCl, 10.7% NaCl, or distilled water 10 min before killing

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 10-4 to 10-12 M in increments of 10-2 M on [3H]tryptophan binding to hepatic nuclei. The results of four experiments indicated that each of the added NaCl concentrations appeared to have an inhibitory effect (%inhibition) on [3H]tryptophan binding: 10-4 M NaCl, 16.0 ± 6.4%; 10-6 M NaCl, 22.0 ± 7.9; 10-8 M NaCl, 21.8 ± 3.0%; 10-10 M NaCl, 20.4 ± 6.1%; and 10-12 M NaCl, 16.8 ± 4.5%. At the same time, the addition of excess (10-4 M) unlabeled L-tryptophan inhibited binding by 67.3 ± 1.8%.

Whether small changes in concentration of added NaCl (between 10-4 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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Fig. 1.   Effect of various concentrations of unlabeled L-tryptophan (star ) or NaCl () on [3H]tryptophan binding to rat hepatic nuclei. Each point represents the mean ± SE of the no. of experiments in parentheses.

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 × 10-5, 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; 10-4 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 (10-4 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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Table 2.   Effects of NaCl when added with unlabeled L-tryptophan on [3H]tryptophan binding to hepatic nuclei

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 10-4 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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Table 3.   Effects of other salts on in vitro [3H]tryptophan binding to hepatic nuclei

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 (10-4 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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Fig. 2.   Saturation isotherm for [3H]tryptophan binding to rat hepatic nuclei in vitro using unlabeled L-tryptophan (10-4 M) without (star ) or with (0.125 × 10-4 M; ) added NaCl. [3H]tryptophan used ranged from 1.25 to 100 nM.

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 (10-4 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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Fig. 3.   Time curve for in vitro [3H]tryptophan binding to rat hepatic nuclei in presence of added distilled water (open circle ), NaCl (0.125 × 10-4 M; triangle ), or unlabeled L-tryptophan (10-4 M; ). cpm, Counts/min.

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 (10-4 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%.

In another group of experiments, we investigated whether preincubation of nuclei with NaCl or unlabeled L-tryptophan for 30-60 min with or without removal of test compounds would affect the subsequent in vitro [3H]tryptophan binding to hepatic nuclei for 1 h (Table 4). The findings with the additions of unlabeled L-tryptophan (10-4 M) or NaCl (10-4, 0.25 × 10-4, and 0.125 × 10-4 M) for 1 h and then adding [3H]tryptophan for 1 h were similar to those obtained in the standard 2-h incubation experiments (Fig. 1). Groups in which hepatic nuclei were incubated with water, unlabeled L-tryptophan, or NaCl for 0.5 h and then spun down, washed, and resuspended in water or NaCl for 0.5 h and then to which [3H]tryptophan was added for the last 1 h are summarized in Table 4. Nuclei pretreated with NaCl or with unlabeled L-tryptophan for 0.5 h and then with the removal of the test compound maintained the subsequent inhibitory nuclear binding effects.

                              
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Table 4.   Effects of additions of L-tryptophan or NaCl on in vitro [3H]tryptophan binding to hepatic nuclei

Kinetics of release of [3H]tryptophan binding. In several experiments, the effect of added NaCl (0.125 × 10-4 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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Table 5.   Effect of addition of NaCl on release of in vitro [3H]tryptophan bound to hepatic nuclei

Free tryptophan levels in hepatic nuclei. To determine whether incubation with varying levels of NaCl (10-5 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 (10-4 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).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 × 10-4 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 (10-4 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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 278(6):C1237-C1245
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society




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