1 Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506; and 2 Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Muscle-stripped piglet colon was
used to evaluate changes in short-circuit current
(Isc) as an indicator of anion secretion. Mucosal exposure to Escherichia coli heat-stable (STa) or
heat-labile enterotoxins (LT) stimulated Isc by
32 ± 5 and 42 ± 7 µA/cm2, respectively.
Enterotoxin-stimulated Isc was not significantly affected by either 4,4'-diaminostilbene-2,2'-disulfonic acid or CdCl2, inhibitors of Ca2+-activated
Cl channels and ClC-2 channels, respectively.
Alternatively,
N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), a compound that inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl
secretion,
reduced Isc by 29 ± 7 and 34 ± 11 µA/cm2, respectively. Two additional diarylsulfonylurea
(DASU)-based compounds were evaluated for their effects on
enterotoxin-stimulated secretion. The rank order of potency for
inhibition by these three closely related DASU structures was identical
to that observed for human CFTR. The degree of inhibition by each of
these compounds was similar for both STa and LT. The structure- and
concentration-dependent inhibition shown indicates that CFTR mediates
both STa- and LT-stimulated colonic secretion. Similar
structure-dependent inhibitory effects were observed in
forskolin-stimulated rat colonic epithelium. Thus DASUs compose a
family of inhibitors that may be of therapeutic value for the
symptomatic treatment of diarrhea resulting from a broad spectrum of
causative agents across species.
cystic fibrosis transmembrane conductance regulator; diarrhea; pharmacology; LY-295501
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INTRODUCTION |
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CONSIDERABLE SOCIOLOGICAL and economic cost is associated with intestinal hypersecretion. Secretory diarrhea ranks as one of the world's top infectious killers, affecting mostly children in third world countries (1) and causing an estimated 5 million human deaths annually (33). In the United States, children under the age of 5 years experience 20-35 million bouts of diarrhea every year, resulting in 2-3.7 million doctor visits, >200,000 hospitalizations, and 500 deaths (12, 20). Although significant focus might be placed on neonatal and pediatric diarrhea, >50% of diarrhea-related deaths in the United States occur in persons over the age of 74, a growing portion of the population (19). Thus significant incentive is present to develop safe, effective, and broad-spectrum treatments for diarrhea.
Vibrio cholera and Escherichia coli are among the major agents responsible for infectious diarrhea in both humans and animals (14). Enterotoxins of E. coli are classified as either heat stable (STa and STb) or heat labile (LT) (13). Cholera toxin (CT) is virtually identical to LT in both structure and function (29, 34). These toxins are not cytotoxic, but they interact directly with intestinal epithelial cells to stimulate profound fluid and electrolyte secretion into the lumen of the gut. Severe dehydration compromises multiple physiological systems in affected individuals and can ultimately lead to death or can set the individual up for opportunistic infection by other organisms.
The biochemistry of enterotoxins and the cellular pathways that contribute to enterotoxin stimulation of electrolyte transport are relatively well characterized (see Refs. 5, 13, 15, 29, and 34 for review). A key player in the response is thought to be the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane anion channel. Treatments for diarrhea aimed at blocking this ion channel have been theoretically proposed (7). Indeed, attempts have been made to reduce toxin-stimulated secretion by blocking anion channels (10), although the compounds available proved not to be effective. The widespread and successful use of oral rehydration therapy in humans indicates that simply circumventing or preventing dehydration will greatly reduce morbidity and mortality associated with enterotoxigenic infections. Thus an intervention that acutely reduces enterotoxin-stimulated intestinal fluid loss will be of great value in both human and veterinary medicine.
Results from the present study document that LT and STa
functionally share a common anion conductive component in porcine colon. Pharmacological evidence indicates that the channel most likely
responsible for enterotoxin-induced Cl secretion in
neonatal porcine colon is the CFTR Cl
channel, which is
blocked by diarylsulfonylureas (DASUs). Comparative results are
presented for the inhibition of rat colonic secretion. Thus
pharmacological intervention to selectively inhibit CFTR is a means by
which a broad spectrum of secretory diarrheas might be managed across species.
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MATERIALS AND METHODS |
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Tissue acquisition. Twenty-seven mixed-breed suckling 7- to 11-day-old pigs were purchased from reputable local sources for use as tissue donors for in vitro studies. Pigs were euthanized by an overdose of pentobarbital sodium in accordance with protocols approved by the Kansas State University Institutional Animal Care and Use Committee. Female Sprague-Dawley rats were anesthetized and then euthanized by cervical dislocation. The spiral colon was removed from each pig, linearized, and flushed with ice-cold Ringer's solution of the following composition (in mM): 120 NaCl, 25 NaHCO3, 1.2 MgCl2, 1.2 CaCl2, 3.3 KH2PO4, and 0.8 K2HPO4. Indomethacin (50 µM; Sigma Chemical, St. Louis, MO) was included in all solutions to preclude prostaglandin synthesis. Distal rat colon was prepared by the same method. In all cases, the colon was split along the mesenteric margin and the muscularis was carefully separated from the epithelial mucosa.
Apparatus. Mucosal epithelium was mounted in modified Ussing chambers (model DCV9, Navicyte, San Diego, CA) with 0.64 cm2 of exposed surface area. Mucosal and serosal compartments contained 5 ml of Ringer solution with 10 mM mannitol and glucose, respectively. Chambers were maintained at 39°C and continually mixed with a bubble lift system (95% O2-5% CO2) that maintained pH at 7.4. Tissues were clamped to zero transepithelial voltage (model 558C, Department of Bioengineering, University of Iowa, Iowa City, IA), and the short-circuit current (Isc), which represents the algebraic sum of active ion-transport processes of the tissues, was recorded. Electrical conductance was determined using Ohm's law by exposing the tissues to a 1-mV bipolar pulse (5-s duration) at 100-s intervals and recording the current deflections. Isc was digitally acquired using an MP100A-CE interface and AcqKnowledge software (version 3.2.6; BIOPAC Systems, Santa Barbara, CA) on a Macintosh computer (Apple Computer, Cupertino, CA).
General protocol. After mounting, tissues were allowed to acclimate for 5-10 min before the transepithelial voltage was clamped. Once a stable baseline was observed, TTX (1 µM; Sigma Chemical) was added to the mucosal chambers to eliminate the residual activity of any nerves remaining associated with the mucosa. Amiloride (10 µM; Sigma Chemical) was then added to the apical compartment to reduce variation between tissues caused by electrogenic Na+ absorption. Tissues were then exposed to either STa (200 ng/ml; Sigma Chemical) or LT (2.5 µg/ml; Sigma Chemical) via the apical solution and allowed adequate time to develop a secretory response. For LT, tissues from 10 pigs were used with the period of exposure ranging from 99 to 259 min (196 ± 16 min) before the addition of putative antagonists. The response to STa was much more rapid, reaching a stable plateau in <20 min. Forskolin, vasoactive intestinal polypeptide (VIP), and serotonin were used as stimulants of Isc in a limited number of experiments. Various selective pharmacological agents were then used to identify cellular components responsible for anion secretion.
Chemicals. 4,4'-Diaminostilbene-2,2'-disulfonic acid (DNDS) was purchased from Acros Organics (Fairlawn, NJ). Forskolin (Coleus forskohlii) was purchased from Calbiochem (La Jolla, CA). E. coli STa and LT, TTX, cadmium chloride, bumetanide, and carbamylcholine were purchased from Sigma Chemical. The DASUs were generously provided by Lilly Research Laboratories (Indianapolis, IN) or were synthesized de novo. All other chemicals were reagent or USP grade. Stock solutions were prepared as follows. Indomethacin (50 mM) and forskolin (10 mM) were dissolved in ethanol; DNDS (5 mM), STa (50 µg/ml), and LT (500 µg/ml) were suspended in Ringer solution; CdCl2 (300 mM) was in water; and DASUs (300 mM) were dissolved in DMSO. For the experiments described here, amiloride, LT, STa, and CdCl2 were added only to the apical compartment, TTX and bumetanide were added only to the basolateral compartment, and all other compounds were added to both compartments.
Data analysis.
Numerical results are reported as means ± SE with the tissue in a
single Ussing chamber as the experimental unit with the following
exceptions. Summary data for basal tissue resistance, basal
Isc, and the effects of TTX and amiloride are
reported using the pig as the experimental unit. Statistical analysis,
including paired t-tests, was completed with Microsoft Excel
(version 8.0; Microsoft, Redman, WA). Treatment effects were considered
to be statistically significant if P 0.05 for a type I
error. Sigma Plot 2000 (version 6.0; SPSS, Chicago, IL) was used for
graphical presentation of the data.
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RESULTS |
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In nonstimulating (i.e., basal) conditions, porcine spiral colon
epithelium exhibited an Isc of 48.8 ± 6.2 µA/cm2 (mean ± SE; 27 pigs, 110 tissues), which is
consistent with either net anion secretion or cation absorption. Basal
resistance in these tissues was 117 ± 8 · cm2. Treatment of the tissues with
TTX to inhibit the activity of any adherent enteric nerves resulted in
a modest but significant reduction in Isc
(
3.6 ± 0.9 µA/cm2; 24 pigs, 98 tissues). The
average change in Isc caused by the addition of
amiloride was
16.8 ± 5.1 µA/cm2
(n = 24 pigs, 98 tissues), indicating that a portion of
the basal current could be attributed to electrogenic Na+
absorption. Three anion transport inhibitors were used throughout the
study [CdCl2, DNDS, and
N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02)]. CdCl2 and DNDS were consistently without
effect on basal Isc. DASU-02 was sometimes
associated with a reduction in basal Isc (see,
e.g., Fig. 4), although statistical significance was not achieved
(P > 0.06) with observations on four tissues.
LT-induced secretion is selectively inhibited by DASUs.
Depicted in Fig. 1 are results from
experiments demonstrating that LT stimulates porcine colonic
Isc in a manner consistent with anion secretion
and that this secretion is selectively sensitive to anion conductance
blockers. As expected, TTX and amiloride reduced
Isc. LT was added to the apical compartment and,
after a delay of 1 h, caused a slowly mounting increase in
Isc. An increase in current to 95 µA/cm2 was observed after an additional 80 min. Anion
channel blockers were then evaluated for their effects on LT-stimulated
Isc. CdCl2, an inhibitor of ClC-2
channels, and DNDS, an inhibitor of outwardly rectifying and
Ca2+-activated Cl channels, were virtually
without effect. Alternatively, DASU-02, a DASU that was previously
reported (26, 27) to inhibit CFTR Cl
channels, caused an immediate and significant reduction in
Isc. It should be noted that the order of
addition had no impact on the outcomes; CdCl2 and DNDS were
without effect and DASU-02 caused profound inhibition. Bumetanide, a
loop diuretic that inhibits intestinal Cl
secretion by
blocking the loading step at the basolateral membrane, was then added
to determine the magnitude of anion secretion remaining. A modest,
although statistically significant, inhibition was observed. Data from
a total of 12 similarly treated tissues are summarized in Fig.
1B. The data show that, compared with the previous
conditions, amiloride (P
0.02), LT (P
0.001), DASU-02 (P
0.005), and bumetanide (P
0.003) significantly altered Isc, whereas
TTX, CdCl2, and DNDS were without effect.
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STa-induced secretion is selectively inhibited by DASUs.
Depicted in Fig. 3 are results from
experiments demonstrating that, like LT, STa stimulates colonic
Isc in a manner consistent with anion secretion
and that this secretion is selectively sensitive to anion conductance
blockers. Again, TTX and amiloride reduced basal
Isc. STa was added to the apical compartment
and, without delay, caused an increase in Isc.
Anion channel blockers were then evaluated for their effects on
STa-stimulated Isc. DNDS and CdCl2
were without effect. Alternatively, DASU-02 caused an immediate and
significant reduction in Isc. It should again be
noted that the order of addition had no impact on the outcomes;
CdCl2 and DNDS were without effect and DASU-02 caused
profound inhibition. Bumetanide again had only a modest effect on
Isc after inhibition by DASU-02, indicating that
little Cl secretion remained. Data from a total of 14 tissues (7 pigs) are summarized in Fig. 3B. The data show
that, compared with the previous conditions, TTX (P
0.003), amiloride (P
0.001), STa (P
0.001),
and DASU-02 (P
0.001), significantly altered
Isc, whereas CdCl2 and DNDS were
without effect.
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DASU-02 precludes enterotoxin-induced anion secretion.
Experiments were conducted to test the hypothesis that DASU-02 could be
effective as a prophylactic treatment for enterotoxin-induced secretion. Data presented in Fig. 4 show
that DASU-02, but not DNDS and CdCl2, can reduce basal
anion secretion. It should be noted, however, that such a reduction in
Isc was not consistently observed. Subsequent
exposure to STa resulted in an increase in Isc,
but the maximal change in Isc in the presence of
DASU-02 was <40% of that observed in control tissues
(n = 2 pairs of tissues from 2 pigs). There was no
difference in response to STa between control tissues and tissues
pretreated with DNDS and CdCl2 (not shown). Virtually
identical results were obtained when VIP was used as the stimulant;
maximal response in the presence of DASU-02 was <20% of that observed
in control tissues or tissues pretreated with DNDS and
CdCl2 (n = 2 pairs of tissues from 2 pigs).
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Structure dependence of DASU inhibition.
Data presented in Fig. 5
demonstrate that inhibition of enterotoxin-stimulated ion transport by
DASUs is structure dependent. All tissues were pretreated with TTX and
amiloride before stimulation with either LT (Fig. 5, A,
C, E, and G) or STa (Fig. 5,
B, D, F, and H). Data from
three to five similar experiments for each set of conditions are
summarized for LT- and STa-stimulated tissues in Fig. 5, I
and J, respectively. Concentration dependence of Isc inhibition for the benchmark compound
DASU-02 is presented in Fig. 5, A and B. It
should be noted that the secretory effects of both LT and STa are
significantly inhibited by 30 µM DASU-02, the lowest concentration
tested (P 0.053 for LT; P
0.048 for STa; n = 5 each); the concentration of 300 µM is
maximally effective, as evidenced by the fact that increasing the
concentration from 300 to 600 µM had virtually no effect. These data
demonstrate a similar concentration dependence for inhibition of both
LT- and STa-stimulated secretion, further documenting that a common target is being affected in tissues stimulated by the two enterotoxins. Typical control data are presented in Fig. 5, G and
H. The solvent vehicle DMSO had no effect on
enterotoxin-stimulated Isc, although nearly
complete inhibition of enterotoxin-stimulated secretion was achieved in
these tissues by the addition of 300 µM DASU-02. LY-295501 is a
structurally related compound that is reported to be oncolytic and is
in Phase II clinical trials. Like DASU-02, LY-295501 reduced
Isc in enterotoxin-stimulated tissues, although statistically significant inhibition (P < 0.05) was
observed only at the highest concentration used (Fig. 5, C
and D). The maximal magnitude of inhibition was similar to
that observed with DASU-02, and little inhibition was observed when
tissues were exposed to DASU-02 in the presence of LY-295501. In
contrast, exposure to CH3-DASU-H, which differs from
DASU-02 only by the para-substituent of the urea phenyl (H vs.
CF3), resulted in virtually no inhibition of
enterotoxin-stimulated Isc, even at the highest
concentration tested (300 µM; Fig. 5, E and F).
Subsequent exposure to DASU-02 resulted in profound and immediate
reversal of the enterotoxin-stimulated increase in
Isc, ruling out the possibility that the tissues
had become unresponsive to DASUs. The rank order of potency
(DASU-02 > LY-295501 > CH3-DASU-H) and the
relative proportion of inhibition were identical, regardless of whether
the stimulant was LT or STa. DASU-02 was shown to be the most potent
inhibitor of LT- and STa-stimulated colonic secretion, with effects
consistently observed at the lowest concentration tested.
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Structure-dependent inhibition of rat colonic secretion.
Results from experiments using rat colonic epithelium are
presented in Fig. 6. It should be noted
that these results closely parallel the observations presented in Fig.
5. A total of 10 closely related DASU-based structures were evaluated,
and results from the three most instructive compounds are presented
here. In the presence of indomethacin, TTX, and amiloride, basal
Isc was 50 ± 7 µA/cm2
(n = 32 tissues). Forskolin caused an increase in
Isc of 192 ± 10 µA/cm2 that
was subsequently reversed by selected DASUs. Once again, DASU-02 was
the most potent of the compounds tested, with inhibitory effects
observed at the lowest concentration tested, 10 µM. More than 80% of
the forskolin-stimulated increment in
Isc was inhibited by 100 µM DASU-02.
Inhibitory effects were likewise observed when LY-295501 was used as
the antagonist. Modest inhibition was observed in the presence of
30 µM, whereas exposure to 100 µM yielded significant reduction in Isc (P 0.001).
Neither CH3-DASU-H nor the carrier vehicle (DMSO) had any
observable effect on forskolin-stimulated ion secretion in rat
colon. Additional experiments indicated that LY-295501 had no effect on
basal Isc but reduced the subsequent effects of
VIP, serotonin, and forskolin (data not shown). These results further
document that DASUs are effective inhibitors of intestinal secretion
across a broad spectrum of stimulants and across species.
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DISCUSSION |
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The results of this study have two major implications. First, a lead compound for the symptomatic treatment of secretory diarrhea is identified. Second, the results confirm previous indications that STa and LT affect a common final pathway of electrolyte secretion, CFTR.
Enterotoxigenic diarrhea depends on the activity of endogenous host
mechanisms, which culminates in activation of an apical anion
conductance. Thus interruption of the host response would limit fluid
loss resultant from colonization and toxin production. Any required
epithelial component would be a logical target for intervention. Zhang
et al. (38) reported that inhibition of the guanylyl
cyclase cascade precluded the effect of STa on intestinal epithelial
cells. Such an approach provides proof of the concept, but specificity
might be an issue in a clinical setting; either multiple cascades must
be blocked or a common element in all cascades must be identified.
Alternatively, it has been suggested (7, 21) that blockage
of apical anion channels would be a logical therapeutic target. In
support of this proposal are numerous studies (6, 11, 30)
that showed that CFTR /
mice were insensitive to enterotoxins,
indicating that a CFTR channel blocker would render the gut resistant
to enterotoxin stimulation. Such an approach was previously taken by
Forsyth and Gabriel (10), although the compounds available
at the time of their studies did not prove to be effective when used in vivo.
Results presented in this study suggest that a newly identified family
of compounds, DASUs, could prove to be effective in the treatment of
enterotoxigenic diarrhea. The results demonstrate a similar
pharmacological profile of ion-transport inhibition for LT, CT,
forskolin, VIP, and STa. The possibility that receptor antagonism, G
protein inhibition, or inhibition of adenylyl cyclase could account for
the inhibition is ruled out by the spectrum of stimulants used. It
remains possible that DASUs could mediate their effects by modulation
of endogenous kinases or phosphatases. However, it was previously
reported (25, 28) that DASUs reversibly block CFTR
Cl channels in excised membrane patches. A reversible
effect in an excised membrane patch strongly suggests a direct
interaction with the ion channel rather than a regulatory protein. Thus
the results presented indicate that each of these agonists stimulates a
cascade that culminates in the activation of CFTR.
The possibility of other anion conductances contributing to the
enterotoxin-induced secretion was evaluated with the experimental paradigms used here. ClC Cl channels are reported to be
present in a variety of epithelial cells including those of the
gastrointestinal tract (17). At least some members of this
gene family of Cl
channels are blocked by
Cd2+ in the micromolar range (3, 9, 23).
Additionally, ClC-2g channels expressed in Xenopus oocytes
are reversibly inhibited by 300 µM Cd2+ (B. D. Schultz, unpublished observations). Ca2+-activated
Cl
channels (CaCC) and outwardly rectifying
Cl
channels (ORCC) are likewise reported to be present in
a variety of epithelia, including those of the gastrointestinal tract.
Although these channels are not yet fully described at the molecular
level, both classes of Cl
channels are widely reported to
be inhibited by disulfonic stilbenes (2, 4, 18, 22, 32, 36,
37). Thus the complete lack of inhibition by both
Cd2+ and DNDS indicates that ClC, CaCC, and ORCC channels
do not participate in enterotoxin-stimulated anion secretion in the
colon, thus eliminating them as possible targets for therapeutic intervention.
DASUs show promise in the symptomatic treatment of enterotoxigenic diarrhea across species. Because oral rehydration therapy is effective in the treatment of enterotoxigenic diarrhea, it is obvious that antibiotic intervention is not required to treat the disease. Rather, if adequate hydration can be maintained, normal physiological processes can bring about a complete cure. We (25) previously found that DASUs inhibited ion transport in epithelial cells of human colonic origin (T84 cells) and here report their effectiveness in pig and rat colon. Thus DASUs hold therapeutic promise for the treatment of secretory diarrhea across a variety of species.
Sheppard and Welsh (31) first reported that sulfonylureas could inhibit CFTR-mediated anion transport. The compounds that they (31) identified, glibenclamide and tolbutamide, were subsequently shown (24, 35) to directly modulate CFTR channel activity in excised membrane patches. However, these compounds are not viable candidates for the therapeutic treatment of diarrhea because they are widely used as antidiabetic agents and are known to induce hypoglycemia. Alternatively, DASUs do not universally cause hypoglycemia (16) and were thus investigated for effects on CFTR. Preliminary evidence has shown that this family of compounds exhibit structure-dependent inhibition of CFTR channel gating (25). The structure and concentration dependence of CFTR inhibition in excised membrane patches was similar to the inhibition of intestinal secretion demonstrated in the present study. Furthermore, the compounds that were most effective in reducing enterotoxin-stimulated anion secretion are known to have modest (DASU-02) or no (LY-295501) effect on insulin secretion or blood glucose levels (16, 25). More importantly, LY-295501 is currently in Phase II clinical trials as an oncolytic, which indicates that it can be safely administered to humans. This compound can be delivered orally and is expected to exhibit a relatively long half-time of elimination based on observations in other species (8). Because CFTR exhibits little variation in structure across species, it is reasonable to predict that similar therapeutic effects would be observed in all species. Certainly, additional studies will be required to determine the in vivo efficacy. At the very least, a lead structure has been identified that is not antimicrobial and appears to be therapeutic for treatment of diarrhea in a variety of species.
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ACKNOWLEDGEMENTS |
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We thank Matt Lenz for technical support and the Kansas State University Swine Unit for animal care.
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FOOTNOTES |
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This work was supported by United States Department of Agriculture National Research Institute Grant 980-2514 and National Institutes of Health Grant T35-RR07064.
Address for reprint requests and other correspondence: B. D. Schultz, Dept. of Anatomy and Physiology, Kansas State Univ., 1600 Denison Ave., VMS 228, Manhattan, KS 66506 (E-mail: bschultz{at}vet.ksu.edu).
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. Section 1734 solely to indicate this fact.
Received 8 February 2000; accepted in final form 12 May 2000.
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