1 Division of Gastroenterology, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan 48109; and 2 Second Department of Internal Medicine, Wakayama Medical College, 811-1 Kimiidera Wakayama, 640-0012, Japan
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ABSTRACT |
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The
role of nitric oxide (NO) and ATP in the regulation of nonadrenergic,
noncholinergic (NANC) inhibitory transmission in the pylorus remains
unclear. In the presence of atropine and guanethidine, electric field
stimulation induced NANC relaxations in a frequency-dependent manner
(1-20 Hz) in the rat pylorus. NANC relaxations were significantly inhibited by NG-nitro-L-arginine
methyl ester (L-NAME; 104 M). P2X
purinoceptor antagonist pyridoxal
phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 3 × 10
5 M) and P2Y purinoceptor antagonist
reactive blue 2 (2 × 10
5 M) had no effect on NANC
relaxations. However, the combined administration of L-NAME
and PPADS, but not reactive blue 2, evoked greater inhibitory effects
on NANC relaxation than that evoked by L-NAME alone.
-Chymotrypsin and vasoactive intestinal polypeptide antagonist did
not affect NANC relaxations. ATP
(10
5-10
3 M) and P2X
purinoceptor agonist
,
-methyleneadenosine 5'-triphosphate (10
7
10
5 M), but not P2Y
purinoceptor agonist 2-methylthioadenosine 5'-triphosphate (10
7
10
5 M), induced muscle relaxations in
a dose-dependent manner, and relaxations were significantly reduced by
PPADS and unaffected by TTX. These studies suggest that NO and ATP act
in concert to mediate NANC relaxation of the rat pylorus. ATP-induced
relaxation appears to be mediated by P2X purinoceptors
located on smooth muscle cells.
P2X purinoceptors; P2Y purinoceptors
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INTRODUCTION |
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IT HAS BEEN DEMONSTRATED that the pyloric sphincter plays an important role in regulating gastric emptying (37, 42, 43). Nonadrenergic, noncholinergic (NANC) innervation is mostly inhibitory in the pyloric sphincter, thus playing an important role in mediating relaxation of the pylorus (39). Previous studies have shown that nitric oxide (NO) biosynthesis inhibitors delay gastric emptying in rats (36) and in dogs (30). A high density of NO synthase (NOS)-immunopositive nerve cells and fibers has been demonstrated in the pylorus (3, 12, 39). It has been demonstrated that in infantile hypertrophic pyloric stenosis, NOS activity of the pylorus is significantly reduced (44). These observations suggest that NO regulates NANC relaxation in the pylorus (2, 24).
Other putative neurotransmitters, such as purinergic (39) and peptidergic neurotransmitters (1, 33), have also been proposed to be NANC inhibitory neurotransmitters in the pylorus. However, the relative contribution of ATP, vasoactive intestinal polypeptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP) in the mediation of NANC relaxations is not fully understood. It has been demonstrated that NOS colocalizes with ATP or VIP in the myenteric plexus (5, 46), but the nature of the interaction between these inhibitory neurotransmitters remains unclear.
Previous studies demonstrated that NO inhibits ACh release in the gastrointestinal tract (19, 27). We have recently demonstrated that NO inhibits the release of VIP and ACh in the rat gastric myenteric plexus (17). Selemidis and colleagues (38) suggested that NO inhibits the release of an apamin-sensitive neurotransmitter in the guinea pig taenia coli. Because apamin is a Ca2+-dependent K+ channel inhibitor and not a selective purinoceptor antagonist, it remains unclear whether there is an interaction between NO and ATP release in the myenteric plexus. In this study, we investigated the possible roles of NO, ATP, VIP, and PACAP as NANC neurotransmitters in the rat pylorus. We also examined the possible interaction between NO and ATP in regulating pyloric relaxations.
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MATERIALS AND METHODS |
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Materials.
The following chemicals were used: ATP, atropine, carbachol,
-chymotrypsin, guanethidine, PACAP, sodium nitroprusside (SNP), TTX, and VIP (Sigma Chemical, St. Louis, MO);
,
-methyleneadenosine 5'-triphosphate (
,
-Me-ATP),
2-methylthioadenosine 5'-triphosphate (2-MeS-ATP),
NG-nitro-L-arginine methyl ester
(L-NAME), L-pyridoxal
phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and reactive
blue 2 (Research Biochemicals, Natick, MA); VIP antagonist
[p-chloro-D-Phe6,Leu17]VIP
(Bachem, Torrance, CA).
Methods. Male Sprague-Dawley rats (body wt 230-250 g) were fasted overnight and euthanized by decapitation under an anesthetic of xylazine and ketamine (13 and 87 mg/kg body wt, respectively). After laparotomy, the stomach and proximal duodenum were removed en bloc and an incision was made along the lesser curvature of the stomach. Circular muscle strips were isolated from the antrum, pylorus, and duodenum. The muscle strips from the antrum and duodenum were obtained 0.5 cm proximal and 0.5 cm distal from the pylorus, respectively. As previously described (39), muscle strips (10 × 2 mm) were suspended under a load of 1 g between two platinum electrodes in an organ bath filled with Krebs-Henseleit buffer of the following composition (in mM): 118 NaCl, 4.8 KCl, 2.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4, and 11 glucose. The Krebs-Henseleit buffer was continuously gassed with 95% O2-5% CO2 and maintained at 37°C and pH 7.4. Mechanical activity was recorded on a polygraph by means of isometric transducers.
Electric field stimulation (EFS; 75 V, 1 ms, 1-20 Hz, 30 s) was applied through two platinum wire electrodes. To determine whether EFS acts through neural pathways, the effects of TTX (10Statistical analysis.
The reduction of the tone induced by EFS or various agonists was
measured and expressed as the relaxation. In the frequency-response studies, the results are expressed as a percentage of the maximal relaxation in response to EFS. In the concentration-response studies, the results are expressed as a percentage of relaxation induced by SNP
(105 M). All data are expressed as means ± SE, and
the number of preparations are reported. Statistical analysis was
performed using ANOVA. Differences were considered significant if the
P value was <0.05.
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RESULTS |
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Contractile patterns of the muscle strips obtained from the antrum,
pylorus, and duodenum.
After an equilibration period of 60 min, almost all of the muscle
strips obtained from the rat pylorus developed much stronger phasic
contractions compared with the muscle strips obtained from the antrum
and duodenum (Fig. 1). The frequency of
spontaneous phasic contractions of the pylorus was 2.5 ± 0.1 cycles/min (n = 5) and the mean contractile force of
each contraction was 11.9 ± 0.8 g/g wet tissue (n = 5). Carbachol (106 M) induced phasic contractions in
the antrum (80.5 ± 7.2 g/g wet tissue; n = 3),
pylorus (21.4 ± 0.7 g/g wet tissue; n = 5), and
duodenum (48.0 ± 1.2 g/g wet tissue; n = 3) (Fig.
1). Thus the pylorus exhibits a contractile pattern different from that exhibited by the antrum and duodenum.
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Effect of L-NAME on NANC relaxations induced by EFS.
In the presence of atropine (106 M) and guanethidine
(10
6 M), EFS (75 V, 1 ms, 1-20 Hz, 30 s)
induced pyloric relaxation in a frequency-dependent manner. These
relaxations were fast in onset and were often followed by off
contractions (Fig. 2A). TTX
(10
6 M) completely abolished relaxations induced by EFS,
suggesting neural mediation. L-NAME (10
4 M)
transiently increased the basal tone by 2.3 ± 0.5 g/g wet tissue
(n = 5) and significantly reduced relaxations induced
by EFS (n = 5, df = 1,40, F = 51.6, P < 0.0001; Fig. 2, B and
C). L-Arginine (10
3 M) completely
prevented the inhibitory effects of L-NAME on EFS-induced NANC relaxation (Fig. 2D).
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Effects of PPADS and reactive blue 2 on NANC relaxations induced by
EFS.
Neither PPADS (3 × 105 M) nor reactive blue 2 (2 × 10
5 M) affected basal tone or inhibited NANC
relaxations induced by EFS (1-20 Hz) (Fig.
3, A and B).
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Effect of combined administration of L-NAME and
purinoceptor antagonists on NANC relaxations induced by EFS.
In the presence of L-NAME (104 M) and PPADS
(3 × 10
5 M) together, the relaxant responses to EFS
were significantly reduced compared with the inhibitory effects of
L-NAME (10
4 M) alone (n = 4, df = 1,30, F = 350.4, P < 0.0001) (Fig.
3A). The combined administration of L-NAME
(10
4 M) and PPADS (3 × 10
5 M) reduced
NANC relaxations in response to EFS (5 Hz) by 69.0 ± 3.3%
(n = 5), a more significant inhibitory effect than that of L-NAME (10
4 M) alone, which was 23.7 ± 4.4% (n = 5, P < 0.0001) (Fig. 3, A and C). In contrast, the combined
administration of L-NAME (10
4 M) and reactive
blue 2 (2 × 10
5 M) did not further inhibit
EFS-induced NANC relaxation compared with the inhibition observed with
L-NAME (10
4 M) alone (Fig. 3, B
and C).
Effect of -chymotrypsin and VIP antagonist.
To determine whether VIP or other neuropeptides are involved in NANC
relaxations in response to EFS of the rat pylorus, the effects of VIP
antagonist (5 × 10
6 M) and
-chymotrypsin (2 U/ml) on NANC relaxations were studied.
-Chymotrypsin (2 U/ml) is a
proteolytic enzyme and has been shown to inhibit NANC relaxations as
well as VIP-induced and PACAP-induced muscle relaxations in the cat
lower esophageal sphincter (29) and rat jejunum
(28). We have previously shown (40) that the VIP antagonist
[p-chloro-D-Phe6,Leu17]VIP
(10
6 M) inhibits NANC relaxations in the rat stomach.
However, the administration of
-chymotrypsin (2 U/ml; data not
shown) and VIP antagonist (5 × 10
6 M; Fig.
4A) had no inhibitory effect
on NANC relaxations induced by EFS in the rat pylorus. Combined
administration of L-NAME (10
4 M) and VIP
antagonist (5 × 10
6 M) did not further inhibit
EFS-induced NANC relaxation compared with the effect produced by
L-NAME (10
4 M) alone (Fig. 4B).
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Effect of exogenous application of SNP, ATP, ,
-Me-ATP and
2-MeS-ATP.
Exogenously applied SNP (10
7-10
5 M),
ATP (10
5-10
3 M), and
,
-Me-ATP
(10
7-10
5 M) induced pyloric
relaxations in a dose-dependent manner (Fig. 5, A and B). In
contrast, 2-MeS-ATP (10
7-10
5 M) had no
effect (Fig. 5, A and B). TTX (10
6
M) did not affect SNP-, ATP- and
,
-Me-ATP-induced muscle
relaxations of the rat pylorus, suggesting that these chemicals act
directly on smooth muscle cells. PPADS (3 × 10
5 M)
significantly inhibited the relaxations evoked by ATP
(10
4 M) and
,
-Me-ATP (10
6 M) by
72.8 ± 6.8% and 75.6 ± 4.4% (n = 3),
respectively. In contrast, reactive blue 2 (2 × 10
5
M) did not affect relaxations induced by ATP (10
4 M) and
,
-Me-ATP (10
6 M). L-NAME
(10
4 M) did not inhibit relaxations evoked by ATP
(10
4 M) and
,
-Me-ATP (10
6 M). PPADS
(3 × 10
5 M) and reactive blue 2 (2 × 10
5 M) did not inhibit relaxations evoked by SNP
(10
5 M; data not shown). Muscle relaxations induced by
ATP (10
3 M) and
,
-Me-ATP (10
5 M) were
not affected by the threshold dose of SNP (10
7 M).
Similarly, SNP (10
5 M)-induced muscle relaxations were
not affected by the threshold dose of ATP (10
5 M) or
,
-Me-ATP (10
7 M; data not shown).
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Effect of exogenous application of VIP and PACAP.
VIP (109-10
6 M) and PACAP
(10
9-10
6 M) have been shown to inhibit
muscle contraction in a dose-dependent manner in various
gastrointestinal tissues in vitro (20, 29). It has been
demonstrated that the threshold dose and ED50 of VIP and
PACAP are 10
9 M and 5 × 10
8 M,
respectively (20, 21, 29). However, VIP
(10
9-10
8 M) and PACAP
(10
9-10
8 M) had no inhibitory effect
on the rat pylorus. Only higher concentrations of VIP
(10
7-10
6 M) and PACAP
(10
7-10
6 M) inhibited phasic
contractions of the rat pylorus (Fig. 6). Inhibitory effects of VIP (10
7-10
6 M)
and PACAP (10
7-10
6 M) were abolished
by pretreatment with
-chymotrypsin (2 U/ml) for 20 min.
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DISCUSSION |
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In the present study, we demonstrated that in the presence of
atropine and guanethidine, EFS (1-20 Hz) caused reproducible relaxations in a frequency-dependent manner. During EFS, the muscle tone was significantly reduced in the rat pylorus. EFS-induced NANC
relaxations were abolished by the pretreatment with TTX, suggesting
mediation by neural pathways. L-NAME (104 M)
significantly reduced EFS-induced relaxations. This suggests that NO is
one of the major inhibitory neurotransmitters in the rat pylorus.
Similar results have been shown in the canine pylorus (2).
A large number of NOS and NADPH diaphorase-positive nerve elements was found in the myenteric plexus of the cat pylorus (3). In anesthetized rabbits, intra-arterial infusion of
NG-nitro-L-arginine
(L-NNA) produced a dose-dependent increase in the frequency
of the pyloric contraction (14). Lingenfelser et al.
(24) demonstrated that both peripheral and central
administration of L-NAME caused enhancement of phasic
pyloric activity in urethan-anesthetized ferrets. These results suggest
that NO acts as an inhibitory neurotransmitter in the mammalian
pylorus. We have previously shown that NANC relaxations evoked by
low-frequency stimulation (<2.5 Hz) were almost completely abolished
by L-NAME (>90%) in the rat stomach (17). As
shown in Fig. 2, A and C, the inhibitory effect
of L-NAME on NANC relaxations evoked by low frequencies
(<2.5 Hz) was smaller (<30%) in the rat pylorus. It is therefore
suggested that, in addition to NO, some other inhibitory
neurotransmitters may be involved in the mediation of NANC relaxations.
It is also conceivable that EFS may release other excitatory
neurotransmitters, such as tachykinins or excitatory ATP, which may
reduce the effectiveness of NO to mediate relaxations of the pylorus.
ATP has also been demonstrated to be an inhibitory neurotransmitter in the gastrointestinal tract (8, 9). However, it remains unclear whether NO and ATP act independently, exerting their effects directly on smooth muscle. In the canine ileocecal junction and the terminal ileum, relaxations induced by ATP are blocked by TTX and L-NNA (6), suggesting that ATP stimulates the release of NO and that NO is the final neurotransmitter mediating muscle relaxation. In our present study, the relaxations induced by ATP were not affected by TTX or by L-NAME, thus excluding the possibility that ATP stimulates the release of NO. It is also unlikely that NO induces ATP release, because PPADS and reactive blue 2 did not affect SNP-induced relaxations of the rat pylorus.
We also demonstrated that PPADS, a P2X purinoceptor antagonist, alone had no inhibitory effect on EFS-induced NANC relaxations of the pylorus. Similarly, reactive blue 2, a P2Y purinoceptor antagonist, did not inhibit NANC relaxation. However, PPADS, but not reactive blue 2, significantly inhibited NANC relaxations in the presence of L-NAME. Because the combined administration of SNP and ATP did not potentiate the relaxations in the rat pylorus, the inhibitory effect of PPADS on NANC relaxations in the presence of L-NAME is not at the muscle level. Conceivably it may involve interaction between neuronal release of ATP and NO.
There is evidence that NO, in addition to its direct action on smooth muscle cells, has an inhibitory effect on neurotransmission in the myenteric plexus. It has been demonstrated that NO inhibits cholinergic transmission (4, 19, 22, 27). Exogenously applied NO donor inhibits the excitatory response to EFS in the rat stomach (17). NO synthesis inhibitors evoke ACh release and enhance EFS-induced contractions in the rabbit stomach (4), guinea pig ileum (22), canine ileum (19), and guinea pig fundus (27). A prejunctional inhibitory effect of NO on substance P neurotransmission has also been demonstrated in the guinea pig ileum (15). We have recently shown that NO inhibits VIP release from the gastric myenteric plexus in rats (17). Furthermore, NO has been suggested to inhibit ATP release from the myenteric plexus (23). Apamin itself has no effect on NANC relaxation, whereas the combination of L-NNA and apamin significantly enhances the inhibitory action of L-NNA on NANC relaxation evoked by EFS in the rabbit internal sphincter (23). We observed a similar phenomenon in the rat pylorus using the P2X purinoceptor antagonist PPADS.
In contrast, Selemidis proposed that the apamin-sensitive neurotransmitter induces relaxation of the taenia coli as well as inhibition of NO release (38). Although L-NNA itself had no effect on NANC relaxations in the guinea pig taenia coli, the combination of L-NNA and apamin significantly inhibited NANC relaxations compared with the inhibitory effects of apamin alone (38). Holzer-Petsche and Moser (16) demonstrated that L-NNA had no effect on NANC relaxation of the rat gastric corpus, but when combined with apamin, L-NNA significantly inhibited NANC relaxations. We propose that a complex interaction involving a prejunctional mechanism may exist between ATP and NO release. The presence of P2X purinoceptors on the nerve terminal of the vagus (35) suggests that ATP may act through a prejunctional mechanism, in addition to directly acting on smooth muscle. It has been shown that nonselective P2 purinoceptor antagonist suramin increases the release of norepinephrine from the sympathetic axons of the mouse vas deferens (45). This suggests that released ATP inhibits subsequent transmitter release via prejunctional P2 purinoceptors. The histological evidence for the coexistence of ATP and NO in the myenteric neurons (5) further supports the possibility of an interaction between these two neurotransmitters at a neuronal level.
It is conceivable that the neural release of NO in response to EFS may inhibit ATP release, in addition to its direct relaxant effects on smooth muscle. Similarly, the neural release of ATP in response to EFS may inhibit NO release via a P2X purinoceptor located on the NO-producing neurons. Administration of L-NAME inhibits the release of NO, which results in removal of inhibitory effects of NO on ATP release. This may explain the inhibitory actions of PPADS on pyloric relaxation in the presence of L-NAME. On the other hand, administration of PPADS alone may block the smooth muscle relaxation via P2X purinoceptor, and PPADS also removes inhibitory effects of ATP on NO release. This potentially explains the lack of action of PPADS on NANC relaxation when it was given alone. However, direct measurement of ATP release and NO release from the myenteric neurons of the rat pylorus is necessary to prove this hypothesis.
There are other possibilities to explain the lack of inhibition of NANC relaxations by PPADS alone, but the combined administration of L-NAME and PPADS evoked greater inhibitory effects on NANC relaxation than that observed with L-NAME alone. It has been shown that P2X purinoceptors are ligand-gated cationic channels that evoke an increase in intracellular Ca2+ concentration from intra- and extracellular stores (10, 48). A low concentration of ATP may activate P2X purinoceptors, causing an increase in intracellular Ca2+ and muscle contractions (10, 48). On the other hand, high concentrations of ATP may cause relaxation as a result of secondary activation of Ca2+-dependent K+ channels. Thus it is possible that the release of ATP (excitatory input) may have contributed to masking the relaxant effects of ATP (inhibitory input). This may account for the fact that PPADS alone seemingly had no inhibitory effect on NANC relaxation. Once the ATP-dependent excitatory inputs are eliminated by PPADS, L-NAME may become a more effective inhibitor of NANC relaxation.
Classically, P2 purinoceptors are subdivided into two
categories, P2X and P2Y. It is generally
accepted that P2X purinoceptors mediate muscle contractions
and P2Y purinoceptors mediate muscle relaxations and/or
suppression of phasic contractions (10). In the blood
vessels, ATP exerts a dual action: relaxation is mediated by
P2Y purinoceptors and contraction is mediated by
P2X purinoceptors (25). It has been
demonstrated that the P2X purinoceptor agonist
,
-Me-ATP causes muscle contraction in the guinea pig ileum
(26) and guinea pig urinary bladder (18),
although recent studies have provided evidence to the contrary. ATP and
,
-Me-ATP induce muscle relaxations that are significantly
antagonized by PPADS in the guinea pig taenia coli (7) and
proximal colon (48). In contrast, ATP and 2-MeS-ATP induce
muscle contractions that are antagonized by reactive blue 2 or suramin
in the guinea pig stomach (31) and in the feline bladder
(41). In our current study, ATP and
,
-Me-ATP induced
relaxations of the pylorus by a TTX-insensitive mechanism, suggesting
that ATP and its analog are acting directly on smooth muscle cells of
the rat pylorus. Muscle relaxations induced by ATP and
,
-Me-ATP
were antagonized by PPADS, but not by reactive blue 2. Therefore, it
appears that ATP induces relaxation through P2X
purinoceptors located on smooth muscle cells of the rat pylorus.
Besides NO and ATP, VIP and PACAP have been shown to be inhibitory NANC
neurotransmitters in various parts of the gastrointestinal tract
(13, 33). However, Soediono and Burnstock
(39) previously showed that VIP (up to 3 × 107 M) did not induce muscle relaxation in the rat
pylorus. Our present study demonstrated that exogenous VIP and PACAP
inhibited pyloric contractions only when high concentrations of these
peptides (>10
7 M) were used. This may indicate the
presence of VIP receptors and PACAP receptors in the rat pylorus, as
previously reported in the pylorus of dogs (1) and rabbits
(33). However, pretreatment with
-chymotrypsin had no
effect on EFS-induced relaxations of the rat pylorus. Furthermore, VIP
antagonist did not affect EFS-induced relaxations. Similarly, Parkman
and colleagues (34) have shown that VIP antagonist was
unable to inhibit NANC relaxations of the rabbit pylorus. Further study
is needed to clarify the possible roles of VIP and PACAP in mediating
NANC relaxations in the rat pylorus.
Combined administration of L-NAME and PPADS, which completely abolished NANC relaxations in response to EFS at low frequency (1 Hz), caused only partial reduction of the NANC relaxations in response to EFS at higher frequencies (5-20 Hz; see Fig. 3B). This indicates that other neurotransmitters may be involved in mediating NANC relaxations in the rat pylorus.
In conclusion, our present study demonstrated that the combination of L-NAME and PPADS, but not reactive blue 2, significantly reduced NANC relaxation of the rat pylorus. Thus ATP and NO released in response to EFS may act in concert to relax the pylorus by some complex mechanisms involving interaction between neural release of ATP and NO.
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ACKNOWLEDGEMENTS |
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This study was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-55808 (T. Takahashi), RO1-DK-39199 (C. Owyang), and P30-DK-34533 (C. Owyang).
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. Owyang, 3912 Taubman Center, Univ. of Michigan Health System, Ann Arbor, MI 48109-0362 (E-mail: cowyang{at}umich.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 December 1999; accepted in final form 4 May 2000.
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