1 Institut National de la Santé et de la Recherche Médicale U45, Hôpital Edouard Herriot, 69437 Lyon Cedex 3; and 2 Laboratoire d'Ecologie et de Physiologie du Système Digestif, Institut National de la Recherche Agronomique, Centre de Recherche de Jouy-en-Josas, 78352 Jouy-en-Josas Cedex, France
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ABSTRACT |
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The hypothesis
that dietary proteins or their hydrolysates may regulate intestinal
mucin discharge was investigated in the isolated vascularly perfused
rat jejunum using an enzyme-linked immunosorbent assay for rat
intestinal mucins. On luminal administration, casein hydrolysate
[0.05-5% (wt/vol)] stimulated mucin secretion in rat jejunum
(maximal response at 417% of controls). Lactalbumin hydrolysate (5%)
also evoked mucin discharge. In contrast, casein, and a mixture of
amino acids was without effect. Chicken egg albumin and its hydrolysate
or meat hydrolysate also did not modify mucin release. Interestingly,
casein hydrolysate-induced mucin secretion was abolished by
intra-arterial TTX or naloxone (an opioid antagonist). -Casomorphin-7, an opioid peptide released from
-casein on milk ingestion, induced a strong mucin secretion (response at 563% of
controls) that was inhibited by naloxone. Intra-arterial
-casomorphin-7 also markedly increased mucin secretion (410% of
controls). In conclusion, two enzymatic milk protein hydrolysates
(casein and lactalbumin hydrolysates) and
-casomorphin-7,
specifically, induced mucin release in rat jejunum. The casein
hydrolysate-induced mucin secretion is triggered by a neural pathway
and mediated by opioid receptor activation.
goblet cells; casein; lactalbumin; -casomorphin; isolated
perfused intestine
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INTRODUCTION |
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A MAJOR FACTOR OF THE FUNCTIONAL barrier of intestine is luminal mucus gel. The mucus coat separates mucosal cells from the exterior milieu and provides protection from noxious substances, allows lubrication of the cell surface, and regulates ion fluxes (10). Mucus, therefore, plays an important role for the intestinal surface integrity in health, and dysfunction of mucus secretion and/or mucin expression could be involved in several pathologies such as inflammatory intestinal diseases and cancer (16, 41). For these reasons, there is a need for better knowledge of the regulation of mucus secretion. In particular, very little is known about the modulation of intestinal mucus secretion by dietary factors, although the interactions of dietary components with the secretory activity of goblet cells could represent new interesting possibilities for the manipulation of this important protective function.
Mucins, the predominant components of the mucus gel, are high-molecular weight glycoproteins with oligosaccharides attached by O-glycosidic bonds to serine or threonine residues on a peptide backbone. In rat small and large intestine, secreted mucins are primarily accounted for by rMuc2 (40) and are mainly synthesised by epithelial goblet cells (or mucus cells) (36). Previous experiments performed either in vivo or ex vivo have demonstrated that dietary fibers and short-chain fatty acids may alter the dynamics of mucus through increased secretion, tissue content, or even altered mucus cell number (2, 18, 27, 28, 35). In contrast, no information on the impact of proteins is available. Dietary proteins and their degradation products are yet involved in the physiological regulation of digestion. In particular, proteins or digested proteins stimulate cholecystokinin release and interact with intestinal endocrine L cells to modulate glucagonlike peptide-1 secretion (4-6, 17). Partial hydrolysates of proteins are also potent stimulants of gastric acid and pancreatic secretion and participate in the regulation of gastrointestinal motility through stimulation of the adrenergic pathway (11, 15, 20). Furthermore, Roberts et al. (26) demonstrated that some dietary peptides may improve wound healing in rats.
The present study was undertaken to determine the effects of dietary proteins or their enzymatic hydrolysates on intestinal goblet cell secretion. For this purpose, we used the preparation of isolated vascularly perfused rat jejunum. This model provides a unique opportunity to study the secretion of mucus in response to well-defined luminal stimuli in a manner that eliminates extraneous influences potentially encountered in vivo. The polarized mucus cells may thus be submitted to specific stimulation with dietary proteins or their products of hydrolysis at a site where nutrient derivatives interact physiologically with the intestinal mucosa. The subsequent jejunal mucin discharge was evaluated by ELISA.
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MATERIALS AND METHODS |
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Materials
BSA was purchased from Biomedia (Boussens, France). Nutrilamine 25, a mixture of amino acids, was obtained from Braun Medical (Boulogne, France). Pancreatic digest of lactalbumin (LH, peptone n°60, ref 20025-037) was from GIBCO-BRL (life Technologies, Cergy Pontoise, France).Animal Model and Experimental Protocol
Surgical preparation. The operative procedure to prepare an isolated vascularly perfused rat jejunum was previously described (7). Briefly, male Wistar rats (250-350 g) purchased from Le Centre d'Elevage Dépré (Saint Doulchard, France) were anesthetized with pentobarbital sodium (50 mg/kg ip). The right and middle colic veins and arteries were tied and cut off near the serosa of the colon. Both ends of the jejunal loop (12 cm in length, 2 cm beyond the ligament of Treitz) were then equipped with Silastic tubing. The jejunal lumen was flushed out with prewarmed isotonic saline and then with air. After the air was gently emptied, both ends of the intestinal loop were ligated. A metal cannula and a Silastic one were then quickly inserted in the superior mesenteric artery and portal vein, respectively. The arterial perfusion started immediately at a rate of 2.5 ml/min with a Krebs-Henseleit buffer [(in mM) 2 CaCl2, 6 KCl, 3.18 NaH2PO4, 104 NaCl, 1 MgSO4, and 41.6 NaHCO3, pH 7.4] containing 25% washed bovine erythrocytes, 3% BSA, 5 mM glucose, and 1% Nutrilamine 25 (vol/vol) and continuously gassed with O2-CO2 (95:5%). The preparation was then removed and transferred to a bath containing isotonic saline at 37°C. The pressure of perfusion, continuously recorded with a mercury manometer, ranged from 40 to 55 mmHg. In preliminary experiments, the viability of the preparation was found to be 1-2 h.
Experimental protocol. The experiments consisted of a 5-min equilibration period followed by a 30-min stimulation period. Immediately after the equilibration period, the loops were filled by injection with 0.8 ml of prewarmed isotonic saline (control preparations or arterial stimulations) or with 0.8 ml luminal factors at 37°C. When required, the pH of luminal compounds to be tested was adjusted to 7-7.5 with diluted hydrochloric acid, and the osmolarity was adjusted to 300 mosmol/kgH2O with sodium chloride. A control experiment was performed for each stimulated loop.
In some experiments, 10Analysis of samples. Samples of luminal contents were incubated for 24 h with 100 mM 1,4-dithiothreitol (DTT) at 4°C for reduction and were then assayed for mucin glycoproteins by an ELISA (see Immunoassay). Samples of luminal stimulants were also assayed for checking the absence of interference in the ELISA.
Jejunal loop homogenates were thawed and briefly homogenized. Aliquots were sonicated for 20 s and then analyzed for DNA content using the fluorimetric method of Hinegardner (14). The amount of mucin glycoproteins secreted from each loop was expressed as micrograms of mucin per milligram of tissue DNA, and the results were expressed as percent controls. Samples of luminal content were also analyzed for DNA content. This analysis served as an indirect measure of tissue viability, and loops were discarded if luminal DNA content represented >2% of the total DNA (tissue DNA + luminal DNA).Purification of Mucin and Development of an Immunoassay
Preparation of rat intestinal mucin. Rat intestinal mucins (RIM) were purified using essentially the same procedure as described for rat colonic mucins (24, 40). Briefly, male Wistar rats were killed by a lethal dose of pentobarbital sodium. Their small intestines were rapidly excised lengthwise and rinsed in cold PBS (4°C). The mucosa was then gently scraped off, and scrapings were homogenized by moderate stirring in the dark for 24 h at 4°C in 50 mM Tris buffer (pH 7.5) containing 6 M guanidinium hydrochloride, protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 10 mM benzamidine hydrochloride, 0.1 mg/ml soybean trypsin inhibitor, and 10 µg/ml pepstatin) and 100 mM DTT. Sulfhydride groups were stabilized by carboxymethylation with 250 mM iodoacetamide and stirring in the dark for 24 h. Insoluble material was removed by centrifugation (30 min, 18,500 rpm, 4°C). Mucins were then purified by equilibrium centrifugation on three consecutive CsCl density gradients (Centrikon T-2055 ultracentrifuge, TFT 50.38 rotor, 40,000 rpm, 70 h, 12°C) as described for rat colonic mucins (24). The high-molecular weight nature of purified mucins was verified by SDS-PAGE and Western blot using an immunological detection system for glycoproteins (DIG Glycan detection kit) (13).
Development of antisera.
Antisera against RIM were obtained in rabbits after repeated injections
of the purified mucins (250 µg). The presence of anti-RIM antibody
was assessed by dot blot. Briefly, serial dilutions (from 6.25 to 200 µg/ml) of purified mucins were blotted on nitrocellulose. Blots were
incubated with anti-RIM (1:1,000 or 1:5,000) and then with goat
anti-rabbit IgG conjugated to horseradish peroxidase (1:1,000). Blots
were developed using the enhanced chemiluminescence method
(chemiluminescence, Pierce, Rockford, IL). The serum with the highest
antibody titer (59C) was selected. On dot blot, no reaction was
observed with bovine albumin, bovine thyroglobulin, bovine
apolipoprotein A-II, bovine apolipoprotein B, human immunoglobulin, bovine glycoprotein -acid, bovine submaxillary mucin, pectin, and
gum arabic. The antiserum 59C was also studied for epitope specificity.
Purified mucins were digested with 0.1 mg/ml proteinase K in 50 mM Tris
buffer, pH 7.4 (1 h, 37°C), to assess the reactivity toward
protease-sensitive epitopes. The digested mucins were blotted on
nitrocellulose and processed as described previously. Proteolytic digestion of the purified mucins with proteinase K resulted in a very
strong decrease in reactivity on dot blot (data not shown), thus
suggesting that protease-sensitive domains in purified mucins were
required for antibody binding.
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Immunoassay. An ELISA for rat intestinal mucin glycoproteins was then established. Wells of a microtiter plate were coated with 100 µl of purified mucins or with sample diluted in carbonate buffer (pH 9.6) and then incubated overnight at 4°C. On the following day, the microtiter plate was washed four times with PBS containing 0.1% Tween (PBS-Tween, pH 7.2). The remaining binding sites in the wells were blocked by addition of 250 µl of PBS-Tween-bovine albumin (0.2 g albumin in 100 ml PBS-Tween) (PBS-Tween-BA) for 1 h at 37°C, and the plate was washed again. At this stage, the wells were incubated with 100 µl of biotinylated-59C (7.5 µg/ml) diluted in PBS-Tween-BA for 1 h at 37°C. After the wells were washed, 100 µl of avidine-peroxidase conjugate were added and allowed to bind for 1 h at room temperature. The plate was washed five times. One hundred microliters of O-phenylenediamine dihydrochloride solution were then added, the color was allowed to develop in the dark for 5-10 min, and the reaction was stopped with 25 µl of 3 M sulfuric acid. The absorbance was read at 492 nm on a microELISA plate reader. A typical standard curve was obtained by using increasing concentrations (0-1,000 ng dry wt/ml) of purified mucins. The curve was linear in a 62.5- to 1,000-ng/ml range. Luminal contents were tested at three dilutions (1:1,000, 1:2,000, and 1:4,000), and all the assays were performed in duplicate. The coefficient of variation of the ELISA was 4.5%. Serial dilutions of luminal samples taken from control or from stimulated loops gave results in ELISA that could be superimposed on the standard curve (data not shown). This immunoreactive material was distributed like purified rat intestinal mucins on a CsCl density gradient, with a peak at a density of 1.4 g/ml, which is characteristic of mucins (40, 42).
Statistical Analysis
Data are presented as means ± SE and were compared using repeated-measures ANOVA followed by paired t-test when appropriate. P < 0.05 was considered significant. ![]() |
RESULTS |
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Effects of Native and Enzymatically Hydrolyzed Dietary Proteins on Mucin Secretion in Rat Jejunum
The casein hydrolysate (CH) we used is a highly refined trypsin hydrolysate from Sigma (ref C-4523). In the isolated vascularly perfused rat jejunum, luminal administration of CH [5% (wt/vol)] induced mucin release (response at 273 ± 54% of control loops, n = 6, P < 0.05). As shown in Fig. 2, the first significant response was observed with 0.1% (wt/vol) CH (277 ± 62% of controls, n = 6), and the maximal response was obtained with 0.5% (wt/vol) CH (417 ± 54% of controls, n = 6).
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In contrast with CH, luminal administration of native casein [5% (wt/vol)] did not significantly stimulate mucin secretion in the isolated perfused rat jejunum. Similarly, neither the amino acid mixture [0.5 and 4% (wt/vol)] nor glutamine (10-100 mM) or glutamic acid (100 mM) induced any discharge of mucin.
Enzymatic lactalbumin hydrolysate [LH; 5% (wt/vol)] led to a
significant increase in luminal mucin content (335 ± 56% of
control loops, n = 5). On stimulation with 0.5%
(wt/vol) LH, the rise in luminal mucin was less pronounced and did not
reach significance (Fig. 3).
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The chicken egg albumin hydrolysate [CEAH; 5% (wt/vol)] only showed a tendency to increase mucin release, and this increase did not achieve statistical significance (140 ± 18% of control loops, n = 6). Administration of 0.5% (wt/vol) CEAH or of native chicken egg albumin [5% (wt/vol)] was also without effect on mucin secretion in rat jejunum.
Luminal administration of an enzymatic meat hydrolysate [5% (wt/vol)] failed to induce any significant increase in the discharge of mucin in rat jejunum (129 ± 17% of control loops, n = 5).
Mechanisms Involved in CH-Induced Mucin Secretion
The effect of the neuronal blocker TTX (10
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Because some casein fragments were shown to behave as opioid receptor
ligands, the effect of intra-arterial infusion of naloxone (antagonist
at µ-, -, and
-receptors) on CH-induced mucin secretion was
investigated. Interestingly enough, mucin secretion induced by 0.5% CH
was inhibited by pretreatment with 10
5 M naloxone.
Naloxone alone, in contrast, was without effect on the mucin secretion
in isolated rat jejunum (response at 116 ± 15% of controls,
n = 6; P > 0.05).
Effect of -CM-7 on Mucin Secretion in the Isolated
Perfused Rat Jejunum
Luminal administration of 1.2 × 104 M
-CM-7
produced a sharp rise in the discharge of immunoreactive material
(563 ± 65% of control preparations, n = 6;
P < 0.05). This concentration of
-CM-7 is
equivalent to what could be obtained from a 0.5% (wt/vol) CH. The
effect of
-CM-7 was concentration dependent over the range 1.2 × 10
5-1.2 × 10
4 M (Fig.
5A), with the first
significant response obtained at 6 × 10
5 M
(response at 300 ± 49% of control loops, n = 6;
P < 0.05).
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As can be seen from Fig. 5B, pretreatment with
105 M naloxone inhibited the
-CM-7-induced mucin
release (response at 146 ± 36% of controls, n = 7 vs. 493 ± 87% of controls for
-CM-7 alone, n = 7; P < 0.05).
On intra-arterial infusion, 1.2 × 106 M
-CM-7
induced jejunal mucin glycoprotein discharge (response at 410 ± 74% of control preparations, n = 7; P < 0.05). On infusion of
-CM-7 at the 6 × 10
7 M
concentration, the rise in mucin discharge was also significant (Fig.
5C).
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DISCUSSION |
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Studies providing direct evidence for intestinal mucus discharge
by nutrients are scarce. Previous investigations showed that dietary
fibers and short-chain fatty acids evoke intestinal mucus secretion
(2, 35). In the present study, we demonstrate, for the
first time, that another class of nutrients might regulate the
secretory function of rat intestinal goblet cells. Indeed, an enzymatic
CH elicited a strong mucin discharge in the rat jejunal lumen. Beside
casein (80%), milk proteins consist of whey proteins (20%)
(30), chiefly lactalbumin, -lactoglobulin, and
immunoglobulins. Our data establish that LH also increased mucin
discharge in the lumen of isolated perfused rat jejunum. Interestingly
enough, the two other dietary protein hydrolysates we tested (meat and chicken egg albumin hydrolysates) failed to elicit jejunal mucin discharge. Thus all protein hydrolysates are not equally potent in
triggering mucin glycoprotein secretion, and mucin discharge in rat
jejunum appears to be critically dependent on the source of dietary protein.
CH produced a dose-dependent release of mucin in the 0.05-0.5% range, with the first significant response at 0.1%. Because the casein content of bovine milk is 2.7%, the CH-induced intestinal mucin secretion may be physiologically relevant not only in the neonate but also in the adult. In this context, it is worth noting that milk proteins constitute the only source of protein for the neonate and up to 20% of a human's average food protein intake in the European community and in the United States (30).
Oligopeptides derived from casein seem specific for mediating the effect of this hydrolysate, because native casein and mixed free amino acids were ineffective to induce mucin release in the isolated rat jejunum. Glutamine is one of the most abundant amino acids in casein and is also the prominent metabolic fuel for small intestinal epithelium. In this study, we show that glutamine did not stimulate mucin discharge. Glutamic acid, the other amino acid tested in this study, was also without effect. These results are meaningful because the enzymatic CH is more likely to mimic dietary protein-derived components found in jejunal chyme than native casein or amino acids.
CH may stimulate intestinal mucin secretion either directly, namely
oligopeptides making contact with the jejunal goblet cells, or
indirectly, e.g., via the enteric nervous system. Because several authors previously showed that intestinal mucus cells are under the
control of the enteric nervous system (22, 24), we
addressed the possibility that this pathway could trigger CH-induced
mucin secretion. In the present study, the CH-evoked mucin discharge was fully blocked by intra-arterial infusion of TTX, thus suggesting the involvement of intramural neurones. The effect of a synthetic analog of casomorphins, -[DAla2,4, Tyr5] CM-5-NH2 on
ion transport in rabbit ileum in vitro was also found to be mediated
neurally (39). A striking finding was that naloxone, an
antagonist at opioid receptors, completely inhibited the mucin release
induced by 0.5% CH. The opioid pathway is thus involved in mediating
the secretion of intestinal mucin produced by CH administration.
The CH used in our experiments is a highly refined trypsin hydrolysate,
and the molecular weights of its oligopeptides are in the
100-1,000 range (informations from the provider). Interestingly, caseins are the source of numerous biologically active peptides such as
opioid agonists, antihypertensive peptides, antithrombotic peptides,
immunostimulants, and mineral carriers. -CMs thus represent the
family of exogenous opioid peptides originally isolated from enzymatic
digests of bovine milk
-casein (37, 38). They are fragments of the
-casein sequence 60-70
(Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu) (21), and
all contain the NH2-terminal amino acid sequence
Tyr-Pro-Phe-Pro.
-CM-5 (fragment 60-64) and -7 (fragment
60-66) were among the first food-derived opioid peptides described
and were found in intestinal chyme or in trypsin hydrolysates
(21). In a final analysis, it can be noticed that
molecular weights of these
-CMs (580 and 790 for
-CM-5 and -7, respectively) are in adequacy with those observed in our CH.
-CMs, first described in the bovine
-casein sequence, are also
found in analogous position in sheep, water buffalo, and human
-casein (21, 25). The well-preserved primary structure of these peptides suggests that
-CMs are important biologically active molecules and raises the question of the significance of these
opioid peptides in diet. In fact, these
-CMs are resistant to the
action of gastrointestinal proteolytic enzymes (25) and could elicit physiological effects in the intestine. Previous studies
showed indeed that
-CMs modulate postprandial release of insulin,
glucagon, somatostatin, and pancreatic polypeptide in dogs, stimulate
intestinal absorption of electrolytes in rabbit ileum, and
significantly prolong intestinal transit time in human and rat
(3, 8, 31-34).
-CMs have also been proposed for the treatment of diarrhea in calves (31). In the present
study, we demonstrate for the first time that
-CM-7 evokes
intestinal mucin secretion in a dose-dependent manner when added to the
luminal side of rat jejunum. Together,
-CMs appear to be important
bioactive peptides in the regulation of intestinal function. Luminal
concentrations of
-CMs after ingestion of milk have not been
determined, but the highest dose of
-CM-7 we used in this study
(1.2 × 10
4 M) is equivalent to what could be
theoretically obtained from a 0.5% CH. In the neonate, such an effect
of milk protein may be a part of the control of mother on defence
mechanisms in the infant's organism, and similar protective effects
may be expected in the adult (29).
The mucin secretion induced by -CM-7 was completely reversed by
naloxone, thus confirming the opioid nature of this effect. Opioid
receptors can be classified into three types (referred to as µ,
,
and
), and
-CMs may represent agonists for µ-receptors detected
in the gut (30, 38). Because numerous
µ-receptor-immunoreactive nerve fibers have been detected around
crypts and blood vessels in the mucosal and submucosal layers of rat
intestine (1), a physiological action of
-CMs requires
the passage of active sequences from the lumen to the basolateral side
of the epithelium. In keeping with this view, intra-arterial
-CM-7
administered at a concentration even 100-fold lower than those used for
luminal administration evoked a sharp mucin discharge in rat jejunum. The mechanism of
-CMs absorption is unknown, but studies with
-lactoglobulin or with insulin (19, 43) support the
hypothesis of a transcytotic transport of peptides through intestinal
enterocytes. The peptides could also be transported intact across
Peyer's patches (9).
It was recently shown that the major whey proteins lactalbumin and
-lactoglobulin also contain bioactive sequences with opioid (lactorphins) or angiotensin I-converting enzyme inhibitory activity (lactokinins) (23). An attractive hypothesis that could
explain mucus secretion induced by LH is the implication of one of
these bioactive peptides. Additional experiments with isolated perfused rat jejunum are thus required to determine which of these peptides is
implicated in mucus discharge induced by the pancreatic digest of
lactalbumin used in our study.
In conclusion, we provide evidence that enzymatic hydrolysates of
casein or lactalbumin may modulate mucin secretion in rat jejunum. In
contrast, native casein, amino acids, chicken egg albumin and its
hydrolysate, or meat hydrolysate does not significantly stimulate mucin
discharge. The secretion of mucin induced by CH or by one of its
well-defined components, -CM-7, is triggered by opioid receptors and
involves a nervous pathway. Our findings extend the list of nutrients
or luminal factors liable to stimulate mucus secretion, which may be of
interest in preventive nutrition and in gastrointestinal pharmacology.
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ACKNOWLEDGEMENTS |
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This work was supported, in part, by the Centre National de la Recherche Scientifique and by the Université Claude Bernard Lyon.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Plaisancié, INSERM U45, Pavillon Hbis, 2ème étage, Hôpital Ed. Herriot, 69437 Lyon Cedex 03, France (E-mail: plaisancie{at}lyon151.inserm.fr).
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.
10.1152/ajpgi.00535.2001
Received 7 January 2002; accepted in final form 20 March 2002.
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