Defective fluid and HCO3minus absorption in proximal tubule of neuronal nitric oxide synthase-knockout mice

Tong Wang1, Fiona M. Inglis2, and Robert G. Kalb2

1 Departments of Cellular and Molecular Physiology and 2 Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Using renal clearance techniques and in situ microperfusion of proximal tubules, we examined the effects of NG-monomethyl-L-arginine methyl ester (L-NAME) on fluid and HCO3- transport in wild-type mice and also investigated proximal tubule transport in neuronal nitric oxide synthase (nNOS)-knockout mice. In wild-type mice, administration of L-NAME (3 mg/kg bolus iv) significantly increased mean blood pressure, urine volume, and urinary Na+ excretion. L-NAME, given by intravenous bolus and added to the luminal perfusion solution, decreased absorption of fluid (60%) and HCO3- (49%) in the proximal tubule. In nNOS-knockout mice, the urinary excretion of HCO3- was significantly higher than in the wild-type mice (3.12 ± 0.52 vs. 1.40 ± 0.33 mM) and the rates of HCO3- and fluid absorption were 62 and 72% lower, respectively. Both arterial blood HCO3- concentration (20.7 vs. 25.7 mM) and blood pH (7.27 vs. 7.34) were lower, indicating a significant metabolic acidosis in nNOS-knockout mice. Blood pressure was lower in nNOS-knockout mice (76.2 ± 4.6 mmHg) than in wild-type control animals (102.9 ± 8.4 mmHg); however, it increased in response to L-NAME (125.5 ± 5.07 mmHg). Plasma Na+ and K+ were not significantly different from control values. Our data show that a large component of HCO3- and fluid absorption in the proximal tubule is controlled by nNOS. Mice without this isozyme are defective in absorption of fluid and HCO3- in the proximal tubule and develop metabolic acidosis, suggesting that nNOS plays an important role in the regulation of acid-base balance.

bicarbonate; acid-base


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE SYNTHASES (NOS) are a family of enzymes that synthesize nitric oxide (NO) from L-arginine in mammalian tissue. Three isoforms of NOS have been identified and defined according to the tissue in which they were discovered: they are present in neurons (nNOS), in endothelial cells (eNOS), and inducible in macrophages and hepatocytes (iNOS) (26, 33). In the kidney eNOS is expressed in renal vascular endothelial cells (36, 37) whereas nNOS is expressed predominantly in the tubule epithelium, Bowman's capsule, especially the macula densa (25, 28, 48), and in principal cells of the collecting duct (47). However, whether nNOS is expressed in the proximal tubule is not very clear. iNOS is widely expressed in the tubule epithelium, including the proximal tubule, thick ascending limbs of Henle's loop, and distal convoluted tubule (27, 37).

In previous microperfusion studies in rats, we have demonstrated that blocking of NOS by NG-monomethyl-L-arginine methyl ester (L-NAME) significantly decreases, whereas luminal application of NO donors such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) increase fluid and HCO3- absorption in the rat proximal tubule. These findings indicate enhancement of fluid and HCO3- absorption by endogenous NO (40). Two important issues remain unresolved. It is not been clear whether NO increases or decreases HCO3- and Na+ transport under physiological conditions, because both stimulation and inhibition of tubule fluid transport have been reported (2, 9, 12, 30, 40). Second, although all three NOS isozymes are expressed in the kidney (1, 3, 22, 36, 37), the physiological role of each isozyme in the regulation of transport has not been defined because selective inhibitors of NOS isozymes are not available. In the present study we examined the effects of L-NAME on urine volume and Na+ excretion and also on fluid and HCO3- absorption in the proximal tubule in control mice. Our data show that L-NAME significantly increased urine volume and Na+ excretion and reduced the rate of fluid and HCO3- absorption in the perfused proximal tubules. However, because L-NAME is a nonspecific NOS inhibitor, these results did not indicate which NOS isozyme(s) play(s) a role in the regulation of fluid and HCO3- transport. In the present study we measured the urinary excretion of HCO3- in mice lacking nNOS, iNOS, and eNOS individually. Because urinary excretion of HCO3- was significantly higher in the nNOS- knockout mice compared with all other groups, we examined the proximal tubule transport of fluid and HCO3- in nNOS-knockout mice.


    METHODS
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INTRODUCTION
METHODS
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Animal preparation and surgical procedures. C57 Black 6J mice were used for studying renal clearances and the effects of L-NAME. Wild-type, iNOS-, and eNOS-knockout mice were obtained from Jackson Laboratory and used for the studies of urine pH and HCO3-. In addition, nNOS wild-type and knockout mice were bred from mice provided by Dr. Paul Huang (Harvard University, Boston, MA) (15). The mice were back-bred on a C57 Black 6J background so that variability within the genetic background of the mice was randomly distributed between (+/+) and (-/-) mice (17). All animals were maintained on a defined diet and tap water until the day of the experiment. The ages of the nNOS-/- animals were matched with their wild-type controls.

Mice were anesthetized by intraperitoneal injection of 100 mg/kg body wt of Inactin [5-ethyl-5-(L-methylpropyl)-2-thiobarbituric acid (BYK-Gulden, Konstanz, Germany)] and placed on a thermostatically controlled surgical table to maintain body temperature at 37°C. After tracheotomy, the left jugular vein was exposed and cannulated with a PE-10 catheter for intravenous infusion. A carotid artery was catheterized with PE-10 tubing for collection of blood and measurement of mean arterial pressure (MAP). The bladder was exposed and catheterized for urine collection via a suprapubic incision with a 10-cm piece of PE-10 tubing.

Renal clearance. Renal clearance studies in mice were carried out as described previously (39, 43). On completion of surgery, 0.3% body wt of isotonic saline was given intravenously to replace surgical fluid losses. Subsequently, a priming dose of 5 µCi of [3H]methoxyinulin (New England Nuclear, Boston, MA) was given in 0.05 ml isotonic saline followed by a maintenance infusion of 0.9% NaCl and 4 mM of KCl containing 10 µCi/ml of [3H]inulin (infusion rate: 0.41 ml/h). An equilibration period of 60 min was allowed before the experiment commenced. The collection periods were 30 min. After two 30-min collection periods L-NAME (3 mg/kg) was given by bolus intravenous (iv) injection. A similar amount of saline was given in a control group. Twenty-microliter blood samples were taken at the beginning and end of each urine collection period. Blood pressure, urine volume, glomerular filtration rate (GFR), absolute excretion rates of Na+ and K+ (ENa, EK), fractional excretion of Na+ and K+ (FENa, FEK), and plasma Na+ and plasma K+ concentrations were determined. In addition, blood pH, PCO2, and HCO3- concentrations were measured by a blood-gas analyzer (Corning Medical and Scientific, Corning, NY). Na+ and K+ concentrations in plasma and urine were measured by standard flame photometry (type 480 flame photometer, Corning Medical and Scientific), and absolute and fractional renal excretion rates were calculated by standard methods (45).

Microperfusion of proximal tubule in vivo. Microperfusion of superficial proximal tubules in vivo was performed by a method similar to that described previously (46). After surgical preparation, the animal was placed on a thermostatically controlled surgical table, and body temperature was maintained at 37°C. Saline solution (0.9%) was infused at a rate of 0.15 ml/h. The left kidney was exposed through a lateral abdominal incision and carefully isolated and immobilized in a kidney cup filled with light mineral oil (37°C). The kidney surface was illuminated by a fiber-optic light. A proximal convoluted tubule with three to five loops on the kidney surface was selected and perfused with a Hampel-type microperfusion pump at a rate of 15 nl/min with a proximal oil block. Tubule fluid collections were made downstream by using another micropipette with a distal side oil block.

The composition of the perfusion fluids was as follows (in mM): 115 NaCl, 24 NaHCO3, 4 KCl, 1 CaCl2, 5 Na-acetate, 5 glucose, L-alanine, 2.5 Na2HPO4, and 0.5 NaH2PO4. The solution was bubbled at room temperature with 5% CO2-95% O2 before use. pH was adjusted to 7.4 with a small amount of NaOH or HCl as required. The perfusion solutions also contained 20 µCi/ml of low-sodium [3H]methoxyinulin for measuring volume absorption and 0.1% FD & C green dye for identification of the perfused loops. After perfusion fluid collections, the perfused tubules were marked with heavy mineral oil stained with Sudan black. To determine the length of the perfused segment, tubules were filled with high-viscosity microfil (Canton Bio-Medical Products, Boulder, CO), the kidney was partially digested in 20% NaOH, and silicone rubber casts of the tubule segments were dissected.

Measurement of rate of volume and HCO3- absorption. The fluid volumes of the original and collected samples were measured in constant-bore glass capillary tubes. The concentration of radioactive [3H]methoxyinulin contained within each sample was determined by liquid scintillation spectroscopy. The rate of net fluid reabsorption (Jv) was calculated according to the [3H]inulin concentration changes between the original and collected fluid.

The HCO3- concentration in the perfusate and collected tubular fluid was measured by using the microcalorimetric method (Picapnotherm) as described previously (41, 46). Collected samples were stored under oil, and the volumes obtained in 15-nl aliquots were compared with sodium bicarbonate standards of 5, 15, 25, and 40 mM. Jv and rate of HCO3- absorption (JHCO3) are expressed per minute per millimeter of proximal tubule.

Statistics. Control and experimental groups were studied under identical experimental conditions. Data are presented as means ± SE. Student's t-test was used to compare control and experimental groups. One-way ANOVA and Dunnett's test were used for comparison of several experimental groups with a control group. The difference between the mean values of an experimental group and a control group was considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of L-NAME on renal clearance. The effects of one dose of L-NAME (3 mg/kg) bolus iv on MAP, GFR, and urine volume are summarized in Fig. 1. The top left panel shows that the baseline of MAP is not significantly different between the control and experimental group before L-NAME administration; however, MAP increased by 28, 26, and 19%, respectively, 30, 60, and 90 min after L-NAME injection, and the average MAP was significantly higher in the L-NAME group. The middle left panel shows that GFR increased slightly after L-NAME administration, but the changes did not reach statistical significance. The bottom left panel of Fig. 1 shows that urine volume doubled after 60 min of L-NAME administration (P < 0.05) compared with the period before administration of L-NAME. We conclude that inhibition of NO synthesis produces a significant diuretic effect in mice.


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Fig. 1.   Left: effects of an intravenous injection of NG-nitro-L-arginine methyl ester (L-NAME; 3mg/kg) on mean blood pressure (BP), glomerular filtration rate (GFR), and urine volume (UV). Right: effects of an intravenous injection of L-NAME (3 mg/kg) on fractional excretion of Na+ (FENa) and K+ (FEK). Data are presented as means ± SE from 8 animals. The control group received saline at the same rate as the experimental group. * Significantly different from control values (P < 0.05).

To investigate the diuretic effects after inhibition of NO synthesis, we examined the effect of L-NAME on urinary Na+ and K+ excretion. As shown in Fig. 1 (top right), FENa significantly increased after L-NAME injection (P < 0.01). FEK did not change significantly (bottom right). These results are consistent with our previous observation that L-NAME significantly increases urine volume and Na+ excretion in the rat (40).

Effects of L-NAME on proximal tubule absorption of fluid and HCO3-. We also investigated the effects of inhibition of NO synthesis on HCO3- and Na+ transport by microperfusion of proximal tubules in vivo. Data are summarized in Fig. 2 and Table 1. L-NAME, given by bolus iv 60 min before samples were collected and added to the luminal perfusion solution (100 µM), significantly decreased both Jv and JHCO3. Jv decreased by 62%, from 1.57 to 0.60 nl · min-1 · mm-1, and JHCO3 decreased by 49%, from 114.0 to 58.0 pmol · min-1 · mm-1, respectively (P < 0.001). The perfusion rates, perfused tubular length, and the HCO3- concentration measured in the original perfusates were similar in the control and experimental groups. These results indicate that inhibition of NO synthesis decreases proximal tubule absorption of fluid and HCO3- directly rather than by a secondary effect due to changes of blood pressure and GFR.


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Fig. 2.   Effects of L-NAME on fluid (Jv) and HCO3- (JHCO3) absorption in proximal convoluted tubule. L-NAME (3 mg/kg) was given by bolus intravenous and was also added to the perfusion solution (100 µM). Data are also presented in more detail in Table 1. * Significantly different from control values (P < 0.05).


                              
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Table 1.   Effects of L-NAME on fluid and bicarbonate absorption in proximal tubules of mice kidney

Proximal tubule HCO3- and fluid absorption in nNOS-knockout mice. Our data show that blocking NOS by L-NAME increases urine volume and Na+ excretion and decreases proximal tubule absorption of HCO3- and Na+. Because all three NOS isozymes are inhibited by L-NAME, our results do not indicate which NOS isozyme(s) is involved in proximal tubule transport. Therefore, we examined the urine pH and HCO3- concentration in nNOS, iNOS, and eNOS-knockout mice. As shown in Fig. 3, urinary HCO3- was significantly higher in the nNOS-knockout animals compared with other groups of mice, although small increases of urine pH and HCO3- were also observed in iNOS-knockout mice and no differences were observed in eNOS-knockout mice. Because nNOS participates more importantly in the control of HCO3- absorption than the other isozymes, we studied the proximal tubule transport of HCO3- and Na+ in nNOS-knockout mice.


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Fig. 3.   Comparison of urine pH and HCO3- concentration in wild-type [endothelial nitric oxide synthase (eNOS)+/+, inducible NOS (iNOS)+/+, and neuronal NOS (nNOS)+/+] and knockout (eNOS-/-, iNOS-/-, and nNOS-/-) mice. Data are presented as means ± SE from 8-10 animals in each group.

Figure 4 is a summary of JHCO3 and Jv in wild-type and nNOS-knockout mice (see also Table 2). JHCO3 and Jv were reduced by 62 and 70%, respectively, in nNOS-null mice compared with the wild-type mice. JHCO3 was 119.3 pmol · min-1 · mm-1 in nNOS+/+ and 44.9 pmole · min-1 · mm-1 in nNOS-/- mice (n = 9, P < 0.01); Jv was 1.48 nl · min-1 · mm-1 in nNOS+/+ and 0.40 nl · min-1 · mm-1 in nNOS-/- mice (n = 9, P < 0.01). These results demonstrate that a large component of HCO3- and Na+ absorption in the proximal tubule is controlled by nNOS.


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Fig. 4.   Fluid and HCO3- absorption rates in wild-type (nNOS+/+) and nNOS (nNOS-/-)-knockout mice. Data are also presented in more detail in Table 2.


                              
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Table 2.   Fluid and bicarbonate absorption in proximal tubules in wild-type and nNOS-null mice

Blood-gas and plasma electrolytes in nNOS-knockout mice. Because proximal tubule JHCO3 and Jv values are significantly lower and urinary HCO3- excretion significantly higher in nNOS-/- compared with nNOS+/+ mice, it is important to know whether the blood pH and HCO3- concentration are affected by this lower JHCO3. As shown in Table 3, the arterial blood HCO3- concentration was reduced from 25.7 to 20.7 mM (P < 0.05) and pH from 7.34 to 7.27 in nNOS-/- mice (P < 0.05). This indicates that animals lacking intact nNOS have a modest but significant metabolic acidosis.

                              
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Table 3.   Acid-base status in wild-type, nNOS-knockout mice and effect of L-NAME

Table 4 summarizes values of mean blood pressure, plasma Na+ and K+ concentrations, and hematocrit. The blood pressure was lower in nNOS-/- mice (76.2 ± 4.6 mmHg) than in wild-type control animals (102.9 ± 8.4 mmHg). The observation that the blood pressure can still rise in response to L-NAME (125.5 ± 5.07 mmHg) demonstrates that L-NAME inhibited the activity of eNOS and/or iNOS in the nNOS-knockout mice. The increments of blood pressure after L-NAME administration are probably caused by vascular constriction inhibition of eNOS activity in the nNOS-knockout mice. It has been demonstrated previously that eNOS-knockout mice develop hypertension (34).

                              
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Table 4.   Blood pressure and plasma electrolytes in control and nNOS-knockout mice


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our previous data have shown that inhibition of NOS by L-NAME increased urine volume and Na+ excretion in rats; L-NAME decreased whereas NO donors enhanced fluid and HCO3- absorption in the rat proximal tubule (40). The present study shows that L-NAME induces similar changes in tubule transport in mice. Second, we show that a significant component of proximal tubule fluid and HCO3- absorption is controlled by nNOS, whereas iNOS may have little effect on HCO3- transport. Third, we observed significant metabolic acidosis in nNOS-knockout mice, supporting the view that this isoform of NOS participates in the regulation of acid-base balance.

It has been suggested that NO is important in the chronic adaptation to increased sodium intake (24, 32, 48) and that NO regulates proximal tubule transport of Na+ (2, 9, 13, 30). However, the reported effects of NO on transport include an increase (2, 9) or a decrease in proximal tubule transport of Na+ and fluid (13, 30). These contrasting NO effects may be due to differences in the renal tubule preparations used (isolated tubules and cells vs. tubules perfused in vivo) or the concentrations of inhibitors or NO donors employed. Indeed, in a previous study we found that low concentrations of NO donors stimulate whereas high concentrations inhibit fluid and HCO3- transport in the proximal tubule (40). The data in our present study show that inhibition of NOS by the nonspecific inhibitor L-NAME significantly decreased fluid and HCO3- absorption in the proximal tubule. These results indicate that endogenous NO increases fluid and HCO3- transport but do not indicate which isozyme is involved in this regulation. For these reasons, use of genetically altered mice offers a unique opportunity to study the role of NO in regulation of kidney tubule transport and to distinguish the important isozyme(s).

In the absence of nNOS, Jv and JHCO3 were significantly lower than in wild-type control mice, indicating that a significant component of fluid and HCO3- absorption is controlled by the action of nNOS. These results parallel the effects of L-NAME, which decreased Jv and JHCO3 in both rat and mouse proximal tubule and support our previous conclusion that endogenous NO enhances fluid and HCO3- transport (40). Other isoforms of NOS, iNOS, and eNOS may play a lesser role in the regulation of proximal tubule transport.

Arterial blood pH and HCO3- were significantly decreased in nNOS-knockout mice, indicating a significant metabolic acidosis. Acute inhibition of NOS by bolus iv of L-NAME had only a transient effect on urine volume and did not significantly affect blood pH and HCO3-. Although the HCO3- absorption in the proximal tubule decreased 60%, the blood HCO3- decreased only 19% in nNOS-knockout mice, similar to the data obtained from Na/H exchanger (NHE3)-knockout mice (31, 46). This suggests a compensatory increase in HCO3- absorption in the late nephron, including upregulation of H+ secretion and K/H exchange. This has also been observed in animals lacking the NHE3 isoform (31). Another possible compensatory mechanism may be a reduction in GFR in nNOS-knockout mice due to activation of tubuloglomerular feedback by the increased delivery of fluid and Na+ to the macula densa. This in turn would be expected to reduce the filtered load of HCO3- and thereby limit the delivery of HCO3- from the proximal tubule, even in the presence of a reduced capacity for HCO3- absorption. Additional free-flow micropuncture experiments are required to investigate the mechanism of this compensation.

Blood pressure was lower in the nNOS-knockout mice, indicating nNOS and eNOS differ in the modulation of blood pressure; eNOS lowers, whereas nNOS raises blood pressure. This conclusion is also supported by the finding that eNOS-knockout animals have high blood pressure (16, 34) and that nNOS-selective inhibitors reduced the blood pressure in eNOS- knockout mice (19). The mechanism of low blood pressure in the nNOS-knockout mice is not clear. Possible explanations include the fact that extracellular volume is reduced by lower tubule absorption of Na+ in nNOS-knockout mice. Indeed, the hematocrit was slightly higher in the knockout mice (48.5 vs. 45.6%), suggesting that plasma volume might be lower in the knockout mice, but these changes did not reach statistical significance.

It is important to know the mechanism of lower Jv and JHCO3 in the absence of nNOS, especially because there is no direct evidence of nNOS expression in the proximal tubule. One possibility is that under physiological conditions, NO produced through the nNOS in Bowman's capsule may go downstream to reach the proximal tubule and decrease endogenous ANG II activity. In the absence of nNOS, endogenous ANG II activity increased. It has been suggested that NO represents a physiological antagonist of ANG II in both the glomerulus and tubule (6, 9, 35). This view is supported by the observation that ANG II blockers prevented the decrease in both single-nephron GFR and proximal tubule absorption during inhibition of NOS (9) and NO donors abolished the stimulation of fluid uptake by luminal ANG II (11). The effects of ANG II on proximal tubule HCO3- and Na+ transport are dose dependent: low concentrations (10-12 to 10-11 M) stimulate, whereas high concentrations (10-8 to 10-5 M) inhibit Na+ and HCO3- transport (14, 41, 42). It is possible that in the mice lacking NO, the activity of ANG II increased, or the local ANG II concentration increased, to reach a level high enough to produce inhibition of proximal tubule transport. Indeed the ANG II concentration in proximal tubule fluid is in a range of 10-8 to 10-9 M, at the border between the stimulatory and inhibitory level (29).

The second possibility is that there is an abnormality in renal nerve development and the adrenergic nerve activity decreased in the absence of nNOS (18). It has been reported that nNOS might play a role in the development of renal catecholaminergic nerves (23). In addition, it has been shown that nNOS expressed in the kidney is associated with nerve bundles (21, 22) and cortical tubules in the rat kidney, especially the proximal tubule receive nerve endings (5, 10). Stimulation of renal sympathetic nerves enhances proximal water and Na+ reabsorption (5); renal denervation induces a significant decrease in water and HCO3- absorption in the proximal tubule (20), and stimulation of adrenergic receptors by norepinephrine increases HCO3- and Na+ absorption in the proximal tubule (7, 8).

In summary, we have found that inhibition of NOS by L-NAME decreases fluid and HCO3- absorption in the proximal tubule of mouse kidney. A significant component of fluid and HCO3- absorption is controlled by the effect of nNOS, and defective absorption of fluid and HCO3- in the proximal tubule in nNOS-knockout mice results in metabolic acidosis and hypotension. The present study demonstrates that NO, in addition to its role in the regulation of blood pressure and hemodynamics (38), modulates fluid and HCO3-absorption in the proximal tubule via changes in nNOS activity and also participates in the maintenance of acid-base balance. Further study is needed to investigate whether any clinical diseases with hypotension and/or metabolic acidosis are caused by nNOS mutation.


    ACKNOWLEDGEMENTS

We thank Drs. R. Berliner and G. Giebisch for reviewing the manuscript and providing constructive comments and Leah Sanders for assistance in its preparation.


    FOOTNOTES

Portions of the study were previously published in abstract form (44). This work was supported by National Institutes of Health Grants DK-17433 and RO1-NS-29837.

Address for reprint requests and other correspondence: T. Wang, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06520.

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.

Received 4 February 2000; accepted in final form 23 May 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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