INTRODUCTION

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HIGH CONCENTRATIONS OF UREA are present in the tissues of marine elasmobranches and in the mammalian renal medulla. Urea generally destabilizes biological macro- molecules, altering their structure and function. Such effects are expected to be deleterious. However, the urea-rich tissues also contain high concentrations of certain methylamine compounds, principally trimethylamine N-oxide (TMAO) in elasmobranches (16) and glycine betaine (betaine) and glycerophosphocholine (GPC) in mammalia (1). These methylamines are believed to protect the tissues from urea by stabilizing macromolecules and thus counteracting the actions of urea (15). The two effects are independently additive (6, 15). When the ratio of methylamines to urea is appropriate (often ~1:2), their opposing effects are reported to counteract, preserving macromolecular structure and function.

Numerous examples have been reported where TMAO, betaine, or GPC counteracts changes in the function of purified enzymes caused by urea (2, 13, 16). TMAO and betaine also counteract effects of urea on protein structure, including changes in thermal stability of ribonuclease (5, 15), in the rate and extent of renaturation of lactate dehydrogenase after acid denaturation (15), and in reactivity of thiol groups in glutamate dehydrogenase (15).

Cells in the renal medulla contain two predominant methylamines, betaine and GPC (3). Although betaine counteracts urea in vitro (16) and in tissue culture (14), the level of betaine in the renal medulla (8, 9, 12) and in tissue culture (7) does not correlate with that of urea, whereas the level of GPC does. In fact, betaine actually decreases with high urea. Thus renal cells apparently prefer GPC over betaine for counteracting the stress of urea. The reason for this is not clear. One intriguing hypothesis is that GPC counteracts the effect of molar levels of urea more effectively than does betaine (11). If so, then this is not a general phenomenon. GPC is significantly less effective than betaine at counteracting the increase in K_m for ADP of pyruvate kinase caused by urea (2). However, this single observation does not exclude the possibility that GPC might counteract some other effects of urea much more effectively than does betaine.

Thus, in the present study, we examine an additional system, comparing GPC vs. betaine in counteracting the thermal destabilization of RNase A by urea (15). Although there is no compelling physiological reason to select this or any other particular cellular macromolecule for such studies, we chose RNase A because it had been previously used in a large number of studies of solute effects on protein structure, including counteraction between urea and methylamines (16), so there is a rich background against which to interpret the present results. The RNase A that we used is derived from bovine pancreas, an organ not normally exposed to high urea. However, counteraction does not depend on whether a particular macromolecule is normally exposed to high urea in vivo or not. For example, essentially the same counteraction is observed with enzymes derived from elasmobranch tissues that are exposed to high urea compared with the same enzymes from mammalian tissues not exposed to high urea (15). The counteracting effects of methylamines and urea are believed to be general ones, dependent on changes in hydration of the macromolecules concerned (6, 15). When we (and others), observe such effects in vitro, we infer that they also occur in vivo, explaining the counteraction of harmful effects of high urea by methylamines that has been documented in living cells (14). Although presence and nature of counteracting effects differ between macromolecules (15), such differences are not known to correlate with whether a specific macromolecule is normally exposed in vivo to high urea concentrations, nor is it apparent that any macromolecule is more important physiologically in this respect than any other.

In the present study, we find that GPC and betaine are approximately equally effective in counteracting the destabilization of RNase A by urea.

	METHODS

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Measurement of the thermal stability of bovine type XII-A pancreatic RNase (catalog no. R-5500; Sigma, St. Louis, MO) was based on a previously published method (15) with some modifications from other similar studies (6, 10).

In brief, 1 mg/ml of RNase A was dissolved in a buffer (10) containing 20 mM sodium citrate (no. 0754; Mallinckrodt, Paris, KY), pH 6.0, and 55 mM NaCl. The organic osmolytes urea, TMAO, betaine (Sigma, B-2754), and/or GPC were added, as indicated. The urea (catalog no. 821527; ICN, Aurora, OH) was treated with Bio-Rad AG-501-X8 mixed bed resin (6), and the TMAO (Sigma, T-0514) was treated with Chelex 100 (Sigma C-7901) (6). GPC was prepared from the 1:1 cadmium chloride adduct (Sigma, G-8005). Cadmium chloride was removed from the GPC before each experiment by shaking a dilute solution of the adduct for 1 h with the mixed bed ion-exchange resin (Bio-Rad, AG-501-X8). The GPC was then concentrated by lyophilizing the solution and dissolving the amorphous residue in a small volume of water. The final GPC concentration was originally confirmed both by direct analysis (4) and freezing-point depression (µOsmette, Precision Systems), following which each new batch was checked by freezing-point depression. Completeness of the removal of the cadmium was tested by flame atomic absorption spectroscopy (Allied Analytical Systems), using a cadmium lamp. No cadmium was detected (<2 µM cadmium in 70 mM GPC solution).

Sets of four solutions were degassed by stirring for 15 min under vacuum at room temperature, then placed in tightly stoppered 3-ml quartz cuvettes (Sigma C-9417) in the middle four positions of the temperature-controlled six-cell positioner (model CPS-240A) of a Shimadzu UV-1601 recording spectrophotometer. The temperature of the solutions was increased between 40 and 73°C, stopping for 5 min after the heating block had reached each new temperature before measuring absorbance at 287 nm. In preliminary experiments, we determined that the absorbance reaches a steady value in this time. Also, we directly calibrated the temperature of solutions in the cuvettes in each position over the range of temperatures used.

The absorbance was read first at 40°C, then successively at each higher temperature up to 73°C, and finally at 40°C again to test for reversibility. The average reversibility was 95% of the difference in absorbance between 40° and 73°C. Absorbance was plotted against temperature for each solution, and the midpoints of the thermal transitions were determined graphically (see Fig. 1). When the four cuvettes were filled with identical control solutions, the mean T_m (temperature of the midpoint of the thermal transition) was identical for all four (63.3°C). Two lots of RNase (Sigma R-5500) were used. The control T_m was slightly higher (63.29 ± 0.01°C, Figs. 1-3) with lot 115H7040 than with lot 86H7046 (62.88 ± 0.10°C, Figs. 4 and 5).

To test whether GPC is degraded during the temperature excursions employed in the experiments, we measured before and at the end of an experiment the osmolality of 0.5 M GPC dissolved in buffer containing RNase A. The osmolality (625 mosmol/kg) did not change, indicating that the GPC is stable. Note that any important hydrolysis of GPC results in increased osmolality as a result of the accumulation of the products, which are choline, glycerol, and phosphate.

Statistics. Statistics were calculated with the GraphPad Instat program, using ANOVA and Student-Newman-Keuls multiple comparison test. P < 0.05 was considered significant. Results are presented as means ± SEM (n = number of measurements).

	RESULTS

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Figure 1 illustrates an experiment comparing the effects of 1.0 M urea, 0.5 M GPC, and the mixture of 1.0 M urea with 0.5 M GPC on the thermal transition of RNase A. The mean values of T_m, the temperature of the midpoint of the thermal transition, for three such experiments are given in Fig. 2. With no organic osmolyte added, the control value is 63.3°C, which is comparable to that previously reported (15), namely 62.7°C. Urea reduces T_m, and GPC increases it. Furthermore, T_m is significantly higher with GPC plus urea than with urea alone, directly demonstrating that GPC counteracts the effect of urea.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of 1.0 M urea, 0.5 M glycerophosphocholine (GPC), and a mixture of the two on the thermal transition of RNase A. Tm, the temperature of the midpoint of the thermal transition, is indicated for each condition by the intercept on the abscissa of the vertical line intersecting the midpoint.

As previously reported (15), TMAO also counteracts urea (Fig. 2). Counteraction by TMAO does not differ significantly from that by GPC (Fig. 2). The concentration of TMAO or GPC which is used (0.5 M) is half that of urea (1.0 M), as in earlier study (15). That ratio was originally chosen because it was found to give approximately complete counteraction and it is close to the ratio found in the tissues of elasmobranchs (15, 16). However, that ratio does not counteract completely in the present study. GPC or TMAO (either at 0.5 M) increases T_m by only 1.0°C, which is substantially less than the 3.1°C that urea lowers it, and with the 1:2 ratio T_m is still 1.8°C lower than control. Although this result does not match the generalization, it is not as exceptional as it might seem. Examination of the data in the earlier study (15) shows a similar, albeit somewhat smaller, deviation from the 1:2 ratio.

Betaine increases T_m, but its effect is less than that of TMAO, as previously observed (15), and also is significantly less (by 0.4°C) than that of GPC (Fig. 2). Similarly, although betaine counteracts urea, that effect is significantly less (by 0.5°C) than the effect of GPC (Fig. 2). To further quantify the difference in counteraction of urea between betaine and GPC, we measured T_m with various concentrations of GPC or betaine added to 1.0 M urea (Figs. 3 and 4). Approximately 1.35 M of either GPC or betaine completely counteracts the effect of 1.0 M urea, indicating that the two methylamines are equivalent in this respect.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of urea, trimethylamine N-oxide (TMAO), betaine, and GPC on Tm of RNase A. All differences are significant (P < 0.05) except for (urea + TMAO) vs. (urea + betaine) and GPC vs. TMAO; number of experiments (n) is indicated with each bar.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of various concentrations of betaine added to 1.0 M urea (n = 3). Control Tm is 63.33 ± 0.03°C. Approximately 1.35 M betaine counteracts 1.0 M urea completely. Error bars are not shown where they are smaller than the marker.

Sorbitol, inositol, and taurine accumulate in renal medullas in addition to betaine and GPC when the interstitial osmolality is high (3). Since the combined effect of all the organic osmolytes might differ from their individual effects, we tested a mixture of compatible organic osmolytes in a ratio similar to that found in rat renal medullary cells (8). This mixture contains 100 mM inositol, 50 mM betaine, 50 mM taurine, 50 mM sorbitol, and 250 mM GPC. The effects of 0.5 M (total concentration) of the mixture (Fig. 4) are similar to those of 0.5 M taurine, betaine, or GPC alone (Fig. 3). Thus the mix of organic osmolytes found in renal medullas apparently does not have any special counteracting effect compared with betaine or GPC alone.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effect of various concentrations of GPC added to 1.0 M urea. Control Tm is 62.97 ± 0.18°C. Approximately 1.35 M GPC counteracts 1.0 M urea completely.

	DISCUSSION

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As depicted in Figs. 4 and 5, GPC and betaine are approximately equivalent in counteracting thermal destabilization of RNase A by urea. In our previous study, we found that GPC is slightly less effective than betaine in counteracting the effect of urea to elevate the K_m of pyruvate kinase for ADP (2). Of course, we cannot rule out the possibility that some other macromolecule is stabilized more effectively by GPC than by betaine, and it is conceivable that more efficient protection of some particularly critical macromolecules still might rationalize the preferential accumulation of GPC by renal cells when urea is high. However, this remains to be seen, and the results to this point do not support the hypothesis that GPC is preferred, because it generally is much more effective than betaine in counteracting the effects of urea on enzymes (11).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effects of urea, mixed osmolytes, and urea plus the mixed osmolytes on the Tm of RNase A. Composition of the mixed osmolytes is 100 mM inositol, 50 mM betaine, 50 mM taurine, 50 mM sorbitol, and 250 mM GPC. All differences are significant (P < 0.05).

Although betaine has been shown to counteract injury of living cells by urea (14), direct evidence is lacking as to whether GPC does this. Thus either betaine or urea, alone, greatly inhibits the survival and growth of renal (MDCK) cells, measured as colony-forming efficiency (14). However, when combined, betaine and urea counteract each other, restoring viability. It would be informative to compare the effectiveness of GPC vs. betaine in this regard, but that is not readily accomplished. Betaine is taken up from the extracellular fluid, so its cellular concentration is easily controlled by altering its concentration in the medium. GPC, on the other hand, is a product of phospholipid metabolism, and it is not obvious how specifically to regulate its cellular concentration, independent of urea. Thus we are left with the generalization that counteraction by methylamines is important for protecting renal cells from high urea, but we cannot directly compare the relative effectiveness of GPC vs. betaine in this regard.

There are other possible reasons for the preferential accumulation of GPC, besides more effectiveness in counteracting urea. Buildup of betaine theoretically is more costly than is buildup of GPC. Betaine is accumulated by transport against a steep concentration gradient, which is energetically expensive. GPC, on the other hand, is continuously produced as a normal product of phosphatidylcholine metabolism. High urea increases GPC in MDCK cells by inhibiting the enzyme that degrades GPC (4). Given that the synthesis of phosphatidylcholine and GPC is not altered, increasing cellular GPC by reducing its degradation does not require additional work as accumulating more betaine does.