(Hypertension. 1997;30:753.)
© 1997 American Heart Association, Inc.
Articles |
Role of Ouabain-like Compound in the Regulation of Transmembrane Sodium and Potassium Gradients in Rats
From the Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, and the Department of Human Dry Dock, Sanraku Hospital (K.Y.), Tokyo, Japan.
Correspondence to Atsuo Goto, MD, Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
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Abstract |
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Abstract A major biologically active Na,K-ATPase inhibitor in the mammalian body may be ouabain-like compound. We investigated the potential roles of circulating ouabain-like compound in the regulation of Na+ and K+ homeostasis in terms of Na+ and K+ distribution between the cells and the extracellular fluid (internal balance). First, we developed a population of rats immunized against ouabain to block the action of ouabain-like compound. We measured plasma and intracellular Na+ and K+ concentrations in skeletal muscle and determined Na+ (extracellular-to-intracellular concentration ratio) and K+ (intracellular-to-extracellular concentration ratio) gradients in immune rats. We examined also the ability to respond to hypertonic NaCl load in immune rats. Consistent lower plasma K+ levels and steeper Na+ and K+ gradients were observed in immune rats. K+ handling in response to hypertonic NaCl load was altered, and lower plasma K+ level was maintained in immune rats. Second, we used PST-2238, a newly developed anti-ouabain agent, to block the action of ouabain-like compound and examined its effect on plasma Na+ and K+ concentrations. Chronic administration of PST-2238 significantly lowered plasma K+ levels in rats with subtotal nephrectomy. These findings collectively suggest that ouabain-like compound may determine at least in part the internal Na+ and K+ distribution and the transmembrane cation gradients in vivo in rats.
Key Words: Na,K-ATPase • sodium • potassium • ouabain • sodium pump
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Introduction |
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The Na,K-ATPase is an integral membrane protein responsible for establishing the electrochemical gradient of Na+ and K+ across the plasma membrane of mammalian cells.1 2 The ion gradients formed by this enzyme are necessary for the active transport of essential nutrients into cells, for osmotic balance and cell volume regulation, and for maintenance of the resting membrane potential in excitable cells. Na,K-ATPase is the only known pharmacological receptor for cardiac glycosides such as digitalis. Much evidence suggests that endogenous digitalis-like factors (EDLFs) or Na,K-ATPase inhibitors may exist in the mammalian body, would modulate the Na+ pump activity, and could be involved in the regulation of Na+ homeostasis.3 4 5 6 7 Increased secretion of EDLF, the purpose of which is restoration of extracellular fluid volume via natriuresis, may lead to elevated cytosolic Ca2+ and vasoconstriction that is responsible for the development and perpetuation of hypertension. Recent evidence suggests that a major biologically active EDLF from the human circulation and the bovine hypothalamus is an isomer of ouabain (ouabain-like compound, OLC).8 9 10 Although there is still growing evidence of other potential EDLFs that are obviously not OLC,11 12 13 OLC appears to fulfill many of the criteria required for EDLFs. However, the physiological roles of OLC in mammals remain to be determined.
OLC would act on Na,K-ATPase in both apolar cells (ie, vascular smooth muscle cells) and polar cells (ie, renal tubular cells) and could induce various in vivo responses. Recent findings suggest that OLC may have a hypertensinogenic action. Several investigators have demonstrated that chronic administration of ouabain leads to the development of hypertension in rats.14 15 16 We developed a population of rats immunized against ouabain to evaluate the consequences of prolonged OLC deficiency on physiological and pathophysiological processes and provided evidence for the contribution of circulating OLC to reduced renal mass-saline hypertension in rats.17 A similar report has supported the participation of OLC to the Na+-induced hypertension in Dahl salt-sensitive rats18 In contrast, the role of OLC in Na+ and K+ homeostasis has been controversial. It is generally thought that salt retention and plasma volume expansion trigger the secretion of OLC. This would explain why salt-loaded humans and patients with congestive heart failure have significantly elevated plasma OLC levels.19 However, acute volume expansion in dogs has been shown to have no effect on plasma OLC levels.20 Studies in patients with chronic heart failure failed to reveal a relationship between atrial pressure and plasma OLC.19 Further, intravenous injection of ouabain was not natriuretic in humans.21 Therefore, exact roles of OLC in Na+ and K+ homeostasis remain to be determined.
Previous studies focused on the potential roles of circulating OLC in the regulation of urinary excretion of Na+ and K+ added to the extracellular fluid (external balance). In this study, we tested the hypothesis that OLC can affect transmembrane Na+ and K+ gradients in vivo (internal balance). For this, we first measured extracellular and intracellular cation concentrations and cation handling in rats immunized against ouabain. Second, we examined effects of the administration of PST-2238, a newly developed anti-ouabain antagonist, on extracellular cation concentrations in rats.
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Methods |
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Immunization Protocol and Titer Determination
All procedures followed were in accordance with the guidelines of the Animal Experiment Committee of the University of Tokyo. One hundred twenty male Wistar rats (Douken, Ibaraki, Japan) weighing 200 to 250 g were immunized against ouabain as described previously.17 To render ouabain immunogenic, ouabain was covalently bound to BSA according to the method of Butler with a slight modification. Initially, animals were injected subcutaneously with ouabain-BSA conjugate in an emulsion of Freund’s complete adjuvant (0.4 mL). Animals were boosted after 1 and 2 months again with ouabain-BSA conjugate in an emulsion of Freund’s complete adjuvant (0.2 mL) and titered 2 weeks after the third immunization. For the titer determination, rat serum was diluted 103-fold by 10 mmol/L phosphate buffer. Diluted serum (0.1 mL) was incubated with 0.3 mL of [3H]ouabain (17.1 Ci/mmol, 0.2 pmol, Du Pont–New England Nuclear) for 24 hours at 4°C. Free [3H]ouabain was precipitated with dextran-coated charcoal and bound [3H]ouabain was counted by liquid scintillation. Eighty-four nonimmunized male Wistar rats served as controls. All rats were raised in a room with alternating 12-hour cycles of light and given free access to food and water. All rats were used for experiments soon after the titer determination.
Autoimmunity Validation
The specificity of OLC blockade by active immunization should be
tested in vivo by studying the effect on cardiovascular
responses of injecting ouabain-immunized rats with exogenous ouabain.
However, it was very difficult to observe a consistent
cardiovascular response to intravenous
ouabain in rats, probably because of the delayed sensitivity of rats to
cardiac glycosides. In this study, the effects of IgG fractions on the
contractile response to ouabain were evaluated in thoracic aorta from
male guinea pigs as reported previously.17
Effects of Ouabain Immunization on Plasma Na+ and
K+ Concentrations
We focused on the changes of plasma Na+ and
K+ concentrations in the following four conditions. All
rats were placed on normal rat chow. First, 7 immune rats and 7 control
rats drank 1% saline solution; 3 weeks later, the blood was collected.
Second, 12 immune rats and 6 control rats received twice a subcutaneous
injection of DOCA (20 mg/kg) and drank 1% saline solution; 1
week later, the blood was collected. Third, 17 immune rats and 12
control rats received an intraperitoneal injection
of furosemide (1 mg/kg); on the following day, the blood was
collected. Fourth, 20 immune and 18 control rats were divided into two
groups. Fourteen immune rats and 6 control rats underwent subtotal
nephrectomy. The other 10 immune rats and 8 control rats underwent sham
operation. Subtotal nephrectomy was achieved as described
previously.22 Three days after the operation, all rats
were given 1% saline solution to drink. After 3 weeks, the blood was
collected.
Effects of Ouabain Immunization on Plasma and Intracellular
Na+ and K+ Concentrations in Skeletal
Muscle
We measured extracellular (plasma) and intracellular
Na+ (Na+e and
Na+i, respectively) and K+
(K+e and K+i)
concentrations in skeletal muscle and determined Na+
(Na+e/Na+i) and
K+
(K+i/K+e)
gradients. First, 10 immune and 10 control rats were used soon after
the titer determination. These rats received no intervention. Trunk
blood was collected and skeletal muscles were excised. Second, 8 immune
rats and 8 control rats received twice weekly a subcutaneous injection
of DOCA (20 mg/kg) and drank 1% NaCl solution. Three weeks
later, trunk blood was collected and skeletal muscles were excised.
Effects of Ouabain Immunization on the Response to Hypertonic
NaCl Load
We examined the alteration of internal Na+ and
K+ balance in response to hypertonic NaCl load in immune
rats. Ten immune and 10 control rats received
intraperitoneally 2 mL of 20% NaCl solution as
reported previously.23 Trunk blood was collected 1 hour
after the injection. To obtain baseline values, the blood was collected
in a similar manner from 10 immune rats and 8 control rats without the
administration of 20% NaCl solution.
Effects of PST-2238 Administration on Plasma Na+ and
K+ Concentrations
PST-2238 is a newly synthesized compound that displaces ouabain
from the Na,K-ATPase and was a gift from Prassis Institute di Ricerche
Sigma-Tau S.p.A. (Milan, Italy).24 It has been shown that
PST-2238 at very low doses antagonizes the pressor effect of ouabain by
interfering at the level of the Na,K-ATPase.25 Sixteen
rats underwent subtotal nephrectomy as described
previously.23 A week later, rats were divided into two
groups of eight rats each. One group received daily administration of
PST-2238 (100 µg · kg-1 ·
d-1 by gavage) suspended in 20%
methylcellulose solution, and the other group received administration
of 20% methylcellulose solution. Both groups of rats were given 1%
saline solution to drink. The other eight control rats underwent sham
operation and drank tap water. After 3 weeks, blood was collected.
Electrolyte Measurements
Intracellular Na+ and K+ concentrations
were determined according to the method of Akaike26 and
Katafuchi et al.27 In brief, extensor digitorum longus
muscles were excised from the right leg, blotted with filter paper, and
weighed. They were then placed into 2 mL of 0.5N HNO3 and
left overnight. The extract was diluted with deionized water to 20 mL.
The intracellular cation concentration (Ci,
mmol/L fiber water) was calculated from the total cation
contents of muscles (Cm, mmol/kg wet weight),
plasma cation concentration (Ce, mmol/L),
extracellular space (v, 1/kg wet weight), and dry-to-wet-weight ratio
(dw/ww) as
Ci=(Cm-vCe)/(1-dw/ww+v). The
extracellular space (v) was obtained from the formula relating the
space (y) to the muscle weight (x) with y=659.8/x+2.67.
In all above experiments, the blood was withdrawn under pentobarbital anesthesia (40 mg/kg) from the abdominal aorta, collected into a tube containing heparin (Ca2+ salt), and plasma cation concentrations were determined. Plasma or tissue cation concentrations were measured by flame photometry.
Statistics
Values are presented as mean±SEM. The data were
analyzed by paired Student’s t test and one-way
analysis of variance, followed by Fisher’s protected least
significant difference, using the StatView-J4.02 system. A value of
P<.05 was considered significant.
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Results |
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Titer of Ouabain-Immunized Rats
Among 120 immunized rats, the percentage of bound to total [3H]ouabain at the titer examination was more than 50% in 71 rats, between 30% and 50% in 38, and less than 30% in 11. The last 11 rats were excluded from further study. Eighty-four nonimmune control rats showed no specific binding at all. Antibody titers to ouabain did not appreciably decline for at least 3 months after the third inoculation with the ouabain-BSA complex.
Autoimmunity Validation
IgG fractions obtained from ouabain-immunized rats but not from
nonimmune rats blocked the vasoconstrictive actions of
exogenous ouabain at the concentration (104 nmol/L)
tested, which was much higher than that found in the circulation (data
not shown). It was thought that the rats immunized against ouabain were
chronically devoid of the major effects of circulating OLC.
Effects of Ouabain Immunization on Plasma Na+ and
K+ Concentrations
Because the immunization took more than 3 months, most rats
weighed between 500 and 600 g at the time of experiments. There
was no significant difference in body weight between control and immune
rats in the following experiments (data not shown).
Fig 1 shows the effects of immunization against ouabain on plasma K+ concentrations under four conditions. After 3 weeks of saline drinking, there was no change in plasma Na+ concentration between two groups of rats (142.0±0.8 versus 142.6±1.2 mmol/L, not significant [NS]). In contrast, plasma K+ concentration was significantly lower in immune rats than in control rats (4.58±0.25 versus 5.52±0.16 mmol/L, P<.05; Fig 1A).
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After a week of DOCA-saline administration, no significant change in plasma Na+ concentration was observed in immune rats compared with control rats (138.5±1.7 versus 137.9±1.7 mmol/L, NS). Ouabain immunization caused a significant reduction of plasma K+ concentration (3.37±0.13 versus 4.09±0.18 mmol/L, P<.05; Fig 1B).
On the next day following an intraperitoneal injection of furosemide (1 mg/kg), plasma Na+ concentration was the same in two groups of rats (146.2±1.4 versus 145.8±1.1 mmol/L, NS). Plasma K+ concentration was decreased in immune rats compared with control rats (4.57±0.15 versus 5.03±0.15 mmol/L, P<.05; Fig 1C).
In two groups of rats with sham operation, there was no difference in plasma Na+ concentration (142.0±0.8 versus 142.6±1.2 mmol/L, NS). On the other hand, plasma K+ concentration was reduced in immune rats compared with control rats (4.58±0.25 versus 5.52±0.16 mmol/L, P<.01; Fig 1D).
Similar results were obtained in rats with reduced renal mass. Although subtotal nephrectomy increased plasma Na+ concentration in two groups of rats compared with corresponding groups of rats with sham operation (P<.05 for both), plasma Na+ concentration in immune rats was not different from that in control rats (153.8±2.5 versus 151.7±3.5 mmol/L, NS). Active immunization with ouabain decreased plasma K+ concentration in rats with reduced renal mass (5.65±0.51 versus 4.52±0.22 mmol/L, P<.01; Fig 1D).
Effects of Ouabain Immunization on Plasma and Intracellular
Na+ and K+ Concentrations
Without any intervention, there was a decrease of plasma
K+ concentration in immune rats (control: 4.73±0.10,
immune: 3.91±0.16 mmol/L, P<.01; Fig 2). No significant change in plasma
Na+ concentration was observed (control: 140.6±0.38,
immune: 141.4±0.62 mmol/L, NS). The
Na+i in skeletal muscle was lower in immune
rats (control: 30.9±1.2, immune: 27.0±1.4 mmol/L,
P<.05; Fig 2). No difference in K+i
was detected between two groups of rats (control: 162.6±4.0, immune:
158.4±3.0 mmol/L, NS). The transmembrane Na+
and K+ gradients were both steeper in immune rats
(Na+e/Na+i,
control: 4.6±0.2, immune: 5.4±0.3, P<.05;
K+i/K+e,
control: 34.5±0.8, immune: 40.9±1.4, P<.01; Fig 2).
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After 3 weeks of DOCA-saline administration, plasma K+ concentration was reduced in immune rats (control: 3.67±0.23, immune: 3.08±0.05 mmol/L, P<.05; Fig 3). There was a trend for higher K+i in immune rats (control: 137.8±4.7, immune: 147.5±1.8 mmol/L, P=.06), and the transmembrane K+ gradient was steeper in immune rats (control: 38.1±2.4, immune: 47.9±1.2, P<.01; Fig 3). However, no significant differences in plasma and intracellular Na+ concentrations and transmembrane Na+ gradient were detected between two groups of rats (data not shown).
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Effects of Ouabain Immunization on the Response to Hypertonic
NaCl Load
We examined cation handling in rats immunized against ouabain
after an intraperitoneal injection of 20% NaCl
solution. The changes in plasma cation concentrations after the
injection are shown in Fig 4. In response
to hypertonic Na+ load, we found a significant elevation of
plasma Na+ concentration in both groups of rats after 1
hour (control: from 140.6±0.4 to 145.7±0.7 mmol/L,
immune: from 141.4±0.6 to 144.7±0.9 mmol/L, both
P<.01). By contrast, plasma K+ concentration
increased from 4.73±0.10 to 5.25±0.12 mmol/L only in
control rats (P<.01). In immune rats, plasma K+
concentration did not rise (baseline: 3.91±0.16, after injection:
3.98±0.17, NS), and a lower plasma K+ level was sustained
compared with control rats (P<.05).
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Effects of PST-2238 Administration on Plasma Na+ and
K+ Concentrations
There was no change in plasma Na+ concentration
between two groups of rats with subtotal nephrectomy (PST-2238:
149.5±3.1, vehicle: 143.3±1.6 mmol/L, NS). In contrast,
plasma K+ concentration was significantly lower in rats
treated with PST-2238 than in vehicle-treated control rats (4.33±0.23
versus 5.45±0.13 mmol/L, P<.01; Fig 5). Thus, PST-2238 reduced plasma
K+ concentration to the level in sham-operated rats
(4.43±0.13 mmol/L).
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Discussion |
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In the present study, we focused on the potential roles of circulating OLC in the regulation of Na+ and K+ homeostasis: the distribution of Na+ and K+ between the cells and the extracellular fluid (internal balance). It will be important to document its bioactivity in long-term studies to prove the role of OLC as a regulator of Na+ homeostasis. For this, we developed a population of rats immunized against ouabain. We hypothesized that prolonged OLC deprivation could be attained by immunizing rats against ouabain and effectively producing autoimmune rats. Similar immunization methods have been successfully used to investigate the roles of atriopeptin or renin in rats.28 29 Since IgG fractions obtained from ouabain-immunized rats effectively blocked the vasoconstrictive actions of exogenous ouabain on guinea pig aorta, we consider the rats immunized against ouabain to be chronically devoid of the major effects of circulating OLC.
The Na,K-ATPase in the cell membrane, which pumps Na+ out of and K+ into the cell in a 3:2 ratio, maintains the normal distribution of Na+ and K+ to the different fluid compartments. The change in Na,K-ATPase activity can affect internal cation distribution. The regulation of the internal distribution of K+ must be extremely efficient, since the movement of a small percent of the cell K+ into the extracellular fluid can result in a potentially fatal increase in the plasma K+ concentration. Catecholamines and insulin induce stimulation of K+ uptake in part by activation of the Na,K-ATPase. The inhibition of the Na,K-ATPase by the administration of cardiac glycosides increases the plasma K+ concentration due to reduced K+ uptake into the cells. Severe hyperkalemia results from cardiac glycoside poisoning. The administration of digoxin-specific Fab antibody fragments induces a significant decrease in the plasma K+ concentration by blocking the action of cardiac glycosides.30
Endogenous OLC, if present, would affect plasma K+ concentration in a similar manner. A hypertensive patient with nonazotemic hyperkalemia caused by a disturbance of internal K+ balance, coexistent with an elevated level of serum digoxin-like factor, has been reported.31 Bagrov et al32 have recently observed a significant correlation between plasma K+ concentration and digoxin-like factor in patients with acute myocardial infarction. In agreement with these observations, we found in this study consistently lower plasma K+ concentration in immune rats under all circumstances tested. Circulating OLC would inhibit Na,K-ATPase in a similar manner to exogenous cardiac glycosides and, as a result, K+ leaves and Na+ enters the cell. On the contrary, when the action of circulating OLC is negligible, Na,K-ATPase is stimulated and K+ enters and Na+ leaves the cell. The net effect could be a lower plasma K+ concentration due to a shift of K+ from the intracellular space to the extracellular space.
We next measured transmembrane Na+ and K+
gradients to test more fully the above hypothesis that OLC can control
the normal distribution of Na+ and K+ and can
affect transmembrane Na+ and K+
gradients.1 2 Three 
-isoforms of the Na+
pump have distinct biochemical properties and bind cardiac glycosides
with different affinities in the rat. Na,K-ATPase possessing the
1-isoform is two orders of magnitude less sensitive to
ouabain than that possessing the 2- and
3-isoforms.2 Considering the low levels of
OLC found in the circulation (0.1 to 1.1 nmol/L), it is tempting
to speculate that the physiological effects of OLC
are mediated by the cardiac glycoside-sensitive isoforms
(2- and/or 3-) but not by the ubiquitous
1-isoform. It is generally thought that skeletal muscle
plays a dominant role in buffering plasma K+ because
skeletal muscle contains the largest single pool of K+ and
also the largest pool of Na+ pumps in the body. McDonough
et al33 proposed that the 2-isoform is key
to buffering against changes in extracellular K+ and may
have evolved to satisfy the needs of the organism for this
capability.
Therefore, we selected skeletal muscle to measure Na+i and K+i and calculated transmembrane Na+ and K+ gradients. In two experiments, the K+ gradient (K+i/K+e) was significantly larger in immune rats compared with control rats. Further, Na+i was lower and the Na+ gradient (Na+e/Na+i) significantly steeper in immune rats with no intervention. We assume that K+ enters and Na+ leaves the cell as a result of the stimulation of Na,K-ATPase due to the deficient OLC action, and both Na+ and K+ gradients rose. These observations support the view that circulating OLC may play an important role in the control of internal Na+ and K+ distribution and in the regulation of transmembrane Na+ and K+ gradients.
In our recent study,23 the twofold or threefold rise in plasma OLC level after an intraperitoneal injection of hypertonic NaCl solution suggested a potential role of OLC in the elimination of excess body Na+ with hypernatremia. Therefore, we compared the ability to handle the hypertonic NaCl load in ouabain-immunized rats with that in control rats. In response to Na+ load, we found a significant elevation of plasma Na+ concentration in both group of rats after 1 hour. By contrast, plasma K+ concentration increased only in control rats. This observation is consistent with our previous finding in normal rats.23 The loaded Na+ would shift from the peritoneal cavity to the vascular space down the concentration gradient via the ion-permeable peritoneal membrane. Because this Na+ movement appears to be a slow process, hypertonic peritoneal fluid would pull the water from the vascular space and induce the rise in plasma K+ concentration, at least for a while. In immune rats, plasma K+ concentration did not rise, and a significantly lower plasma K+ level was sustained. Thus, the rats could not normally handle Na+ load without the action of circulating OLC. In the absence of OLC, Na,K-ATPase activity would be stimulated, and K+ would easily shift into the cell from the extracellular space.
We used PST-2238 as an alternative method to block the action of OLC in a long-term study. It has been shown that PST-2238 at very low doses antagonizes the pressor effect of ouabain or OLC by interfering at the level of Na,K-ATPase and prevents blood pressure elevation with chronic ouabain administration or in Milan hypertensive rats.25 We used subnephrectomized rats drinking saline solution because we have observed the rise in plasma OLC levels in this model.22 Chronic treatment with PST-2238 significantly lowered plasma K+ concentration and reduced it to the level in control rats. This preliminary finding suggests that circulating OLC may play an important role in the control of internal Na+ and K+ distribution at least in rats with subtotal nephrectomy.
In summary, chronic blockade of circulating OLC induced lower plasma K+ level, steeper K+ gradient (K+i/K+e), lower Na+i, and steeper Na+ gradient (Na+e/Na+i). Further, K+ handling in response to Na+ loading was altered in the absence of circulating OLC in rats. An anti-ouabain agent also induced significantly lower plasma K+ levels. These findings collectively suggest that circulating OLC may control the distribution of Na+ and K+ via the regulation of plasma membrane Na,K- ATPase activity and determine the transmembrane Na+ and K+ gradients in vivo.
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Acknowledgments |
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This study was supported in part by a grant-in-aid (07457164) from the Ministry of Education, Science, and Culture, Japan. We are indebted to Prof Giuseppe Bianchi and Dr Patrizia Ferrari for the generous gift of PST-2238.
Received March 17, 1997; first decision April 22, 1997; accepted April 30, 1997.
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