Elevated Lymphocyte Cytosolic Calcium in a Subgroup of Essential Hypertensive Subjects
Abnormalities of intracellular calcium homeostasis and sodium-proton exchange have been implicated in the pathophysiology of essential hypertension. To further define the nature of cytosolic calcium abnormalities and whether they relate to increased sodium-proton exchange in hypertension, we have studied peripheral lymphocytes from normotensive and hypertensive subjects. Lymphocyte cytosolic calcium was significantly increased (P<.01) in hypertensive compared with normotensive subjects while consuming a high salt diet. Using maximum likelihood analysis, we found that cytosolic calcium levels in our study population were not normally distributed and observed three modes (P<.02). The means of the first mode and the two upper modes were separated (±2 SD) at a cytosolic calcium level of 120 nmol/L. We conducted further analysis in the subgroups with cytosolic calcium levels >120 nmol/L or <120 nmol/L. The majority of the normotensive subjects (86%) and half of the hypertensive subjects (52%) had levels <120 nmol/L. Clinical characteristics of the two subgroups did not differ. Subjects with levels <120 nmol/L had a rise in cytosolic calcium when changed to a low salt diet; those with levels >120 nmol/L did not show a change in cytosolic calcium but their blood pressure fell significantly with salt restriction. Hypertensive subjects also had increased sodium-proton exchange activity compared with normotensive subjects when both groups were studied in a high salt balance. A positive correlation between sodium-proton exchange and cytosolic calcium was observed in subjects with levels <120 nmol/L. There was insufficient power to draw conclusions on this relationship in subjects with levels >120 nmol/L. Thus, many hypertensive subjects have increased cytosolic calcium, but this abnormality is not associated with sodium-proton exchange activity in all individuals. The salt-induced change in cytosolic calcium in subjects with levels <120 nmol/L and its link to sodium-proton exchange suggest regulation by factors involved in salt-volume homeostasis. Individuals with cytosolic calcium >120 nmol/L, most of whom were hypertensive, may have abnormalities in this regulation, contributing to hypertension.
The important role of cytosolic Ca2+ (Cacyt) in vascular smooth muscle contraction has supported the hypothesis that abnormal intracellular Ca2+ homeostasis may be involved in the hypertensive process. Because of some similarities between platelets and vascular smooth muscle cells, particularly in their ability to respond to vasoactive hormones, platelets are frequently used as a surrogate cell for investigation of changes in Ca2+ homeostasis in vivo. The observation that blood pressure and platelet Cacyt from hypertensive individuals were correlated1 led to further studies in humans.2 3 4 Also, platelets, lymphocytes,5 6 and vascular smooth muscle cells7 from genetic strains of hypertensive rats have elevated Cacyt levels.
Increased Cacyt in platelets from hypertensive subjects is not unique to this blood element. Elevated Cacyt has also been reported in lymphocytes8 9 and erythrocytes10 11 from individuals with essential hypertension. These observations are helpful, as the platelet is not ideal for the study of underlying mechanisms because of its lability with in vitro manipulations. Peripheral lymphocytes are a more stable cell population than platelets or mixed leukocytes.
These abnormalities in basal Cacyt have been postulated to be linked to more widespread changes in intracellular ion regulation that may include modifications in intracellular Na+ and pH.11 Hypertensive individuals have also been reported to have increased Na+-H+ exchange activity in erythrocytes,12 platelets,13 and lymphocytes.14 It has been hypothesized that elevated Na+-H+ exchange activity might be linked to the increased Cacyt levels observed in human hypertension.15 16 However, little experimental evidence has supported this concept.
The present study had three objectives: (1) to carefully define Cacyt levels in peripheral lymphocytes from normotensive and hypertensive subjects, (2) to assess the activity of Na+-H+ exchange in both groups and its relationship to Cacyt levels, and (3) to assess the effect of changes in salt intake on Cacyt. Our results indicate that Cacyt has a multimodal distribution in normotensive and hypertensive subjects, with nearly half of hypertensive subjects having elevated Cacyt levels. Salt restriction significantly increases Cacyt in subjects with levels <120 nmol/L but not in those with higher levels. Na+-H+ exchange activity is also significantly elevated in hypertensive subjects but mainly in those with low Cacyt (<120 nmol/L). In these subjects, a significant positive correlation was demonstrated between Cacyt and Na+-H+ exchange, suggesting that a relationship between these two markers of intracellular ion regulation exists in some hypertensive individuals.
Forty-two subjects with essential hypertension and 36 normotensive subjects were studied. All untreated hypertensive subjects had blood pressure >140/90 mm Hg documented before study participation, and those on antihypertensive medications were withdrawn from medication 3 weeks before enrollment in the study. None of the female subjects was taking estrogen preparations (either postmenopausal estrogen replacement or oral contraceptive medications) within 3 months of the time of study. Menopausal status of the female subjects was not obtained. The protocol was approved by the Human Subjects Committee of the Brigham and Women's Hospital, and informed, written consent was obtained before participation.
Blood samples were obtained from individuals participating in studies investigating the hormonal and cardiovascular effects of dietary salt manipulation. Study subjects were consuming a high salt diet (200 mEq Na+/d), and all measurements were obtained on day 7 of the high salt diet. Blood pressure measurements were obtained with an indirect recording sphygmomanometer (Dinamap, Critikon), and blood samples for biochemical measurements and lymphocyte studies were taken after subjects had been supine for 30 minutes and fasting overnight. A subgroup of 29 subjects was studied during a second week when they consumed a low salt diet (10 mEq Na+/d). Blood samples for lymphocyte studies were again obtained on day 7 as above.
Human lymphocytes were isolated from whole blood as described by Strazzullo and Canessa.17 Briefly, 20 mL of venous blood was collected in tubes containing EDTA and was centrifuged (200g) at room temperature for 15 minutes. After removal of the platelet-rich plasma, the leukocyte-enriched buffy coat and red blood cell pellet were suspended in Ca2+-, Mg2+-free Hanks' balanced salt solution (HBSS) at 10% hematocrit. For preferential selection of lymphocytes, carbonyl iron (0.5 mg/mL) was added to the suspension to be phagocytized by monocytes and other macrophages. This mixture was incubated for 30 minutes in a shaking water bath at 37°C, and the iron-containing cells were precipitated with a magnet. The supernatant was layered on a solution containing 2.5 mL Percoll, 2.0 mL HBSS, and 0.3 mL NaCl (1.5 mol/L) and centrifuged for 40 minutes. The cells were washed and later suspended in HBSS and kept at room temperature. Trypan blue exclusion as a measure of cell viability was typically >80%. Methods for further purification of the cell preparation were not used; thus, this cell preparation is predominantly lymphocytes and not completely devoid of platelets and red blood cells. Similarly, prepared peripheral lymphocytes have been shown to consist of approximately 90% T and 5% B lymphocytes.18
Measurements of Cacyt
Steady-state levels of Cacyt were determined with the fluorescent probe fura 2 as described by Tsien et al19 with some modification. Lymphocytes were incubated with 2 μmol/L fura 2-acetoxymethyl ester (Molecular Probes) in 1 mL physiological saline solution (PSS) containing (mmol/L) NaCl 140, KCl 5, HEPES 10 (pH 7.4), NaHPO4 1, CaCl2 1, MgSO4 0.5, and glucose 5 for 30 minutes at 37°C. The cells were washed twice with 1 mL PSS, and aliquots of 2×106 cells were suspended in prewarmed PSS. Before fluorescence was recorded, the cells were centrifuged, resuspended in fresh buffer, and placed in a cuvette. Fura 2 fluorescence was recorded for 3 minutes at 510 nm emission wavelength, with alternating excitation at 340 and 380 nm with a spectrofluorometer (model 650-10S, Perkin-Elmer). Stable values were obtained for periods up to 10 minutes. Cell fluorescence was corrected for autofluorescence, and the Cacyt concentration was calculated as described.19 Although this method does not correct for dye leakage, given the short residence time of samples in the cuvette and the observation that longer time periods continued to provide stable values, it was felt that the contribution of leakage was minimal, as previously suggested.18 Preliminary experiments performed with Mn2+ to quench extracellular dye showed that <4% of the fluorescence signal was affected over the time period of study. All determinations were performed in duplicate for each subject. Repeated assays (three measurements) were performed in six normotensive and hypertensive subjects for estimation of the reproducibility of these measurements; the coefficient of variation was 10%.
Measurements of Na+-H+ Exchange Activity and Basal pHi
Measurements of Na+-H+ exchange activity (measured as Na+-dependent H+ efflux) were performed as described by Strazzullo and Canessa.17 Lymphocytes loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Molecular Probes) were acid-loaded to different pHi with nigericin (0.5 μg/mL) and suspended in choline chloride-containing medium. The cells were then divided into equal aliquots (50 μL) and sequentially transferred to the spectrofluorometer with fluorescence recording at 530 nm emission wavelength and alternating excitation at 495 and 440 nm. One sample remained in choline medium and was used for confirmation of the acid-loaded pHi as well as buffering capacity as described below. NaCl medium (2 mL of medium containing 140 mmol/L NaCl, pH 7.4) was added to the other 50-μL sample. Both NaCl and choline chloride media were isosmotic; therefore, the final osmolality remained constant. In the presence of NaCl, prompt alkalinization occurred, and the change in pHi was recorded for 30 seconds. The initial rate of alkalinization was calculated from the difference between initial pHi and the pHi recorded 15 seconds after addition of NaCl medium. This time period represents the linear portion of the alkalinization curve. Each sample was measured in duplicate for each condition. Basal pHi was determined as above in cells suspended in PSS (pH 7.4). Intracellular buffering capacity was determined with the pHi titration of acidified cells by measurement of the subsequent change in pHi with addition of 2.5 mmol/L NH4Cl using the method described by Roos and Boron.20
In some experiments, Cacyt was chelated with 40 μmol/L 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA, Molecular Probes), which was added to the suspension after a 10-minute preincubation with BCECF. The cells were then acid-loaded to various pHi values for measurement of alkalinization rates. Cacyt was measured in a separate group of the BAPTA-treated cells as described above.
Blood samples were collected on ice and centrifuged at 4°C, and plasma was stored at −20°C until the time of assay. Aldosterone and plasma renin activity were measured by radioimmunoassay. Plasma and urinary electrolytes were measured with an ion-selective electrode system.
Data are reported as mean±SE unless otherwise stated. ANOVA, Student's t test, the Wilcoxon rank sum test, two-way ANOVA, linear regressions, and the Pearson product moment correlation were performed with the CLINFO software statistical program or SigmaStat. To determine whether the data for Cacyt were best described by a single or multiple distributions, we performed maximum likelihood analyses.21 The null hypothesis was rejected at a value of P<.05.
Clinical characteristics of the two study populations are shown in Table 1⇓. Hypertensive subjects were older and had greater body mass index (BMI) than normotensive subjects. No significant differences between men and women were found in any of these parameters.
Cacyt Levels in Lymphocytes
Cacyt levels were significantly elevated in hypertensive compared with normotensive subjects (130±13 versus 62±5 nmol/L, P<.01). Among the normotensive subjects, Cacyt was higher in men versus women. However, this sex difference disappeared in the hypertensive subjects. Among women, the hypertensive women showed a threefold greater Cacyt level than the normotensive women (Fig 1⇓). Cacyt levels in both male and female hypertensive subjects were significantly higher than in normotensive subjects of the same sex. A weak but significant correlation (P=.03, r=.26) between mean blood pressure and Cacyt in hypertensive and normotensive subjects was observed. Given that age and BMI were different between the groups, we analyzed these parameters to determine their contribution to the Cacyt differences between hypertensive and normotensive subjects. No significant correlation between age or BMI and Cacyt was found in normotensive and hypertensive subjects as a group or when analyzed by sex. Additionally, two-way ANOVA revealed that age and BMI did not significantly contribute to the elevated Cacyt seen in hypertensive subjects.
The frequency distribution for lymphocyte Cacyt in normotensive and hypertensive subjects is shown in Fig 2⇓. Maximum likelihood analysis on this data showed that Cacyt in the normotensive and hypertensive subjects was not normally distributed but was multimodal (P<.02). Day21 has described this method of statistical evaluation of data for determination of the presence of multimodality. In this analysis, the number of observations, separation of the means of the subpopulations, relative size, and SD of the distributions are considered. With this analysis and with separation of modes by 3 SD values (one mode representing 30% of the total observations), only 50 observations are required for documentation of bimodality. Similarly, separation of modes by 0.5 SD would require more than 200 observations. For analysis of these data, equal and unequal SD values of the modes were modeled. With this analysis, the data were best described by three modes, with means (±SD) of 70±24, 150±14, and 246±64 nmol/L. The lowest mode was readily distinguished from the two higher modes given that 2 SDs from the means overlapped at a level of approximately 120 nmol/L. Most of the normotensive subjects (86%) and half of the hypertensive subjects (52%) were within the first mode. The second mode contained 14% of the normotensive and 31% of the hypertensive subjects. The highest mode, which encompassed the entire group >120 nmol/L, contained 48% of the hypertensive and 14% of the normotensive subjects. Therefore, a Cacyt level <120 nmol/L contained nearly all of the normotensive subjects, and a level >120 nmol/L consisted of largely hypertensive subjects. On the basis of these observations, we chose to compare characteristics of subjects with Cacyt >120 nmol/L and <120 nmol/L. The two groups did not differ significantly in their clinical characteristics, except that the group with Cacyt >120 nmol/L had significantly higher blood pressure because of the higher number of hypertensive subjects; this group also had no black subjects (Table 2⇓).
Responses of Blood Pressure and Cacyt to Salt Restriction
Twenty-nine subjects (12 normotensive and 17 hypertensive) had Cacyt measured while they consumed both high and low salt diets. Eleven normotensive subjects and 9 hypertensive subjects had Cacyt <120 nmol/L while on the high salt diet. These subjects had significantly (P<.05) higher Cacyt levels on the low salt diet compared with the high salt diet, but blood pressure did not change with altered salt intake (Fig 3⇓). Cacyt levels did not change with salt restriction in those subjects with Cacyt levels >120 nmol/L; however, mean blood pressure fell significantly in these subjects during the low salt diet (−11±1 mm Hg, P<.05). Both groups of subjects (>120 nmol/L and <120 nmol/L) had appropriate increases in plasma renin activity and aldosterone levels with salt restriction. In particular, the >120 nmol/L group, which demonstrated a fall in blood pressure induced by salt restriction, did not have an overrepresentation of subjects with low plasma renin activity. Plasma renin activity and aldosterone levels did not differ between the groups during either the high or low salt diet (Table 3⇓).
Na+-H+ Exchange Activity in Lymphocytes
To study Na+-H+ exchange activity, we determined Na+-dependent H+ efflux as a function of pHi in both normotensive and hypertensive subjects during the high salt diet. Initial experiments performed in six hypertensive and normotensive subjects showed that Na+-H+ exchange activity had a similar Km in both subject groups, whereas Vmax was higher in the hypertensive subjects. These preliminary experiments also indicated that accurate measurements of Vmax were difficult in subjects with high Na+-H+ exchange activity because rapid recording of pHi changes in <1 minute were not precise. This led us to choose the single level of pHi 6.5 (316 nmol/L intracellular H+) for comparison of Na+-H+ exchange activity in lymphocytes of hypertensive and normotensive subjects.
Na+-H+ exchange activity was determined in lymphocytes from 18 normotensive and 20 hypertensive subjects consuming a high salt diet who were randomly selected from the total study population (Table 4⇓). Na+-H+ exchange activity was significantly higher in hypertensive than normotensive subjects (29±6 versus 16±6 mmol/L per minute, P<.05). Basal pHi was measured in some of the subjects and revealed similar values in hypertensive and normotensive subjects (P=.09).
Relationship Between Cacyt and Na+-H+ Exchange Activity
To assess whether a relationship between Cacyt and Na+-H+ exchange activity exists in vitro, we performed experiments to buffer Cacyt using the membrane-permeable calcium chelator BAPTA. In these experiments, Na+-H+ exchange activity was measured at basal Cacyt and after reduction in Cacyt with BAPTA. Lowering Cacyt from 54 to 20 nmol/L resulted in >90% reduction in Na+-H+ exchange activity measured at pHi 6.5. These data provided suggestive evidence that Cacyt can regulate Na+-H+ exchange. However, to address the question of whether increased Na+-H+ exchange activity in hypertensive subjects is linked to elevation of Cacyt, we measured Na+-H+ exchange activity and Cacyt simultaneously in lymphocytes from normotensive and hypertensive subjects. In the total group, there was no correlation between Cacyt and Na+-H+ exchange activity in lymphocytes. However, when the normotensive and hypertensive subjects with Cacyt <120 nmol/L were analyzed, a significant relationship was evident (Fig 4⇓). Insufficient numbers of subjects had measurements of Na+-H+ exchange activity and Cacyt >120 nmol/L for us to be able to draw conclusions about this relationship, but the data indicate that four of five subjects with Cacyt >120 nmol/L did not have elevated Na+-H+ exchange activity.
In the present study, we measured Cacyt levels in lymphocytes from hypertensive and normotensive subjects under conditions of balanced salt intake. Maximum likelihood analysis of this large sample of normotensive and hypertensive individuals indicated that Cacyt is not normally distributed. Although most normotensive subjects (86%) had Cacyt levels <120 nmol/L, only half (52%) of the hypertensive subjects had similar levels. These data indicate that a substantial number of hypertensive individuals exhibit abnormalities in intracellular Ca2+ homeostasis but that these abnormalities are not shared by all hypertensive subjects. This elevation of steady-state Cacyt levels might be due to decreased efflux or enhanced Ca2+ influx or intracellular mobilization; however, we did not design these studies to identify the site of abnormal Cacyt handling.
Attempts to correlate Cacyt levels and blood pressure have not been universally successful4 although it has been suggested that such a correlation exists.1 3 This could be related to the fact that Cacyt levels in normotensive and hypertensive subjects overlap substantially, as evidenced by the present observations and those of others.1 2 3 4 Within our normotensive population, women had lower Cacyt levels than men. This relationship disappeared in the hypertensive group, but levels in the hypertensive women were threefold greater than in normotensive women. This sex difference is intriguing but without explanation. We did not obtain information on menstrual cycle or menopausal status from our study subjects, so we cannot determine the contribution of these factors. Normotensive women in our study were younger than hypertensive women. In relation to this, blood pressure in premenopausal women is lower than that in men of similar age, and menopause induces an odds ratio of 2.2 for the development of hypertension compared with the premenopausal state.22 Likewise, blood pressure in postmenopausal women becomes equivalent to or higher than that in men of similar age. We also found that elevated Cacyt levels in hypertensive subjects did not correlate with age or BMI. Although each of these variables—sex, age, and BMI—may have a significant effect on the predisposition for developing hypertension, it is apparent that none predicted the presence of elevated Cacyt in the hypertensive population.
A second important finding of this study is that subjects with a Cacyt level <120 nmol/L showed evidence of its regulation by changes in salt intake. The observation that these subjects have reduced Cacyt levels when consuming a high salt versus low salt diet indicates that mechanisms associated with salt-volume regulation are capable of altering Cacyt. These mechanisms do not appear to operate in subjects with levels >120 nmol/L (mostly hypertensive subjects). A significant salt-induced change in blood pressure was observed in this group with high Cacyt as well. Such subgroup analyses, based on the multimodal distribution of Cacyt in our study population, allowed these relationships to be revealed.
Other researchers have shown that salt intake can affect Cacyt levels in hypertensive individuals. Whereas we found that salt intake did not change Cacyt in the subjects with a salt restriction-induced fall in blood pressure, Alexiewicz et al9 and Resnick et al11 showed that Cacyt in salt-sensitive hypertensive individuals rises with high salt diet. Reasons for differences between our results and these previous reports are not readily apparent. However, neither of these studies examined subjects at the extremes of sodium intake used in the current study and neither studied Cacyt in normotensive subjects for comparison.
Discussion of the physiological relevance of these changes in Cacyt as they relate to blood pressure must be considered speculative. However, if such changes reflect similar events occurring in vascular smooth muscle, they may be germane to the control of blood pressure during changes in salt intake. Salt restriction leads to salt depletion and intravascular volume depletion, both of which are reversed with high salt intake. However, few normotensive subjects and no more than 50% of hypertensive individuals manifest “salt sensitivity,” that is, a significant change in mean blood pressure between low and high salt intakes. This lack of a blood pressure response to increased salt intake is related in part to adjustments in vascular tone. A rise in vascular Cacyt with a low salt diet would in principle result in increased peripheral resistance and maintenance of blood pressure in the setting of volume depletion. When these subjects were on a high salt diet, the resulting salt and volume expansion would cause blood pressure to rise were it not for a concomitant reduction in vascular tone as reflected by a reduction in Cacyt. These predicted changes in Cacyt are what we observed in the normotensive and hypertensive subjects with lymphocyte Cacyt <120 nmol/L. In contrast, those subjects with Cacyt >120 nmol/L, who did not adjust Cacyt in lymphocytes (and presumably vascular smooth muscle) in the setting of a change in salt intake, developed a significant salt-induced rise in blood pressure. This hypothesis is in agreement with the observations of Sullivan et al,23 who measured peripheral vascular resistance in salt-resistant and salt-sensitive normotensive and hypertensive individuals during high and low salt diets. They found that salt-resistant subjects demonstrated a significant change in peripheral vascular resistance between high and low salt diets; peripheral vascular resistance was higher during the low versus high salt diet. In contrast, peripheral vascular resistance did not change in salt-sensitive subjects between high and low salt intakes, yet blood pressure rose significantly with salt loading.
We also studied the relationship between Cacyt levels and Na+-H+ exchange activity because both parameters appear to be abnormal in hypertensive subjects. We confirmed that lymphocyte Na+-H+ exchange activity is increased in hypertensive subjects. We also found that a normal basal level of Cacyt is required for maintenance of Na+-H+ exchange activity in lymphocytes. In our subjects, basal pHi was not different between hypertensive and normotensive subjects although a trend was observed. Although not confirmatory, these data are consistent with those of Saleh and Batlle,24 who showed that lower pHi levels are associated with elevated Na+-H+ exchange in the spontaneously hypertensive rat.
In the subgroup of subjects with Cacyt levels <120 nmol/L, Cacyt was correlated with Na+-H+ exchange activity. The Cacyt modulation of this antiporter has been documented in vascular smooth muscle cells,25 fibroblasts,26 red blood cells,27 and platelets.28 The interaction of Cacyt with the exchanger has also been shown to be mediated by the presence of a calmodulin binding site in the NHE-1 isoform of the Na+-H+ exchanger.26 A relationship between Cacyt and Na+-H+ exchange was not seen in subjects with Cacyt >120 nmol/L although this may have been due to insufficient power to detect such a relationship. These latter observations may further suggest that abnormalities of these two markers of intracellular ion regulation are not linked in all hypertensive individuals. The presence of defects in one or both parameters may represent the result of separate and distinct abnormalities.
The present observations provide evidence that Cacyt levels are elevated in lymphocytes of some hypertensive individuals. We identified subgroups within our population that could be divided at a Cacyt level of 120 nmol/L, with most of the normotensive individuals being below this level. In subjects with Cacyt <120 nmol/L, salt restriction resulted in increases in Cacyt levels. Cacyt did not change in response to dietary salt manipulation in those subjects with high Cacyt, but these subjects did show significantly higher blood pressure levels than levels measured during low salt intake. Such findings suggest that Cacyt is affected by factors involved in sodium homeostasis and this regulation is abnormal in hypertensive individuals with high Cacyt. Also, Na+-H+ exchange activity was correlated with Cacyt in subjects with levels <120 nmol/L. These data support the concept that abnormalities of intracellular ion regulation in many hypertensive subjects, particularly Cacyt and Na+-H+ exchange, represent unique phenotypes in certain subgroups of hypertensive individuals. Further study of these subgroups may provide additional information to help in the identification of candidates for structural/genetic abnormalities associated with hypertension.
This research was supported by grants from the National Institutes of Health (NIH) (HL-42120, HL-461071, HL-38655) and a grant from the Department of Veterans Affairs. Dr P.R. Conlin was supported by a Clinical Associate Physician Award from the NIH National Center for Research Resources (RR-02635); A. Rivera was supported by the same grant. This research was conducted in a General Clinical Research Center, and the data were analyzed by a Computerized Database Management and Analysis Systems (CDMAS) facility, both supported by a grant from the NIH National Center for Research Resources (RR-02635). We wish to thank Dr Steven C. Hunt (University of Utah School of Medicine) for performing the maximum likelihood analysis.
Reprint requests to Paul R. Conlin, MD, Endocrine-Hypertension Division, Brigham & Women's Hospital, 221 Longwood Ave, Boston, MA 02115.
- Received October 19, 1995.
- Revision received December 14, 1995.
- Revision received December 14, 1995.
- Accepted March 25, 1996.
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