![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 284, Issue 1, 362-369, 1998
Department of Pharmacy, Division of Pharmaceutics, Uppsala University, Sweden
 |
Abstract |
|---|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
Medium chain fatty acids (MCFAs) are used to enhance the permeability of mucosal tissues to hydrophilic drugs, but their mechanism of action is largely unknown. In this study, the absorption-enhancing effects of the sodium salts of two MCFAs, capric acid (C10) and lauric acid (C12), were studied in monolayers of human intestinal epithelial Caco-2 cells. Both MCFAs induced a rapid increase in epithelial permeability to the hydrophilic marker molecule sodium fluorescein. Inhibition of phospholipase C and inhibition or activation of various kinases and buffering of intracellular calcium indicated that the effects on epithelial permeability were mediated through phospholipase C-dependent inositol triphosphate/diacylglycerol pathways. Surprisingly, the inositol triphosphate and diacylglycerol pathways were found to have opposing effects on paracellular permeability. Exposure to the MCFAs also resulted in a concentration dependent reduction of cellular dehydrogenase activity and ATP levels. C10, but not C12, induced redistribution of the tight junction proteins ZO-1 and occludin. These results indicate that the two MCFAs have partially different and more complex mechanisms than previously recognized, which has important implications for their use in vivo.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
The
sodium salt of the MCFA C10 is used as an absorption enhancer in drug
products marketed in Japan, Denmark and Sweden, but only limited
information is available regarding its mechanism of action (Anderberg
et al., 1993
; Lindmark et al., 1995; Tomita et al., 1995). Morphological studies have demonstrated that
C10 regulates the paracellular permeability of the tight junctions in
Caco-2 monolayers and in rat and human intestinal segments, indicating
that it has a specific effect in the intestinal epithelium (Anderberg
et al., 1993; Sawada et al., 1991;
Söderholm et al., 1995). Recent studies on viable
epithelial cell monolayers using fluorescent marker molecules support
the hypothesis that C10 enhances drug permeability through the tight
junctions (Lindmark et al., 1997b). Inhibition studies
suggested that the effects of C10 are regulated through a phospholipase
C-dependent pathway (Tomita et al., 1995). In general, these
studies were performed after long incubations (>1 hr) with C10, and
limited information is available on its immediate effects on the
intestinal epithelium. This is perhaps surprising because the immediate
effect of an absorption enhancer is likely to be more relevant than the
long-term effect in vivo. When an absorption enhancer
(together with a drug) is released from a formulation in the
gastrointestinal tract, it will be rapidly diluted in the
gastrointestinal fluids. As a result, the absorption enhancer will be
present only in concentrations sufficiently high to increase epithelial
permeability immediately after the release from the formulation. We
therefore also investigated the short-term effects of the MCFAs in the
present study.
A recent investigation identified the sodium salt of the MCFA C12 as
another interesting regulator of epithelial permeability (Lindmark
et al., 1995). In contrast to C10, C12 did not induce detectable changes in tight junction morphology, but
electrophysiological measurements indicated that C12 also specifically
regulates paracellular permeability in the intestinal
epithelium.3 The absence of
morphological changes suggests that C12 has at least a partly different
mechanism of action from C10. However, no information is available on
the mechanism by which C12 regulates paracellular permeability.
In this study, the short- (t = 0-12 min) and long- (t = 12-60 min) term effects of C10 and C12 on epithelial permeability to
hydrophilic model drugs were studied in Caco-2 monolayers. The
simplicity of this cell culture model makes it suitable for studies of
the direct regulation of paracellular permeability in intestinal
epithelial cells (Hochman et al., 1994; Lindmark et
al., 1995; Tomita et al., 1995; Yen and Lee, 1995).
Effects on the energy status of the epithelial cell monolayers as well as effects on intracellular signalling pathways were investigated.
| |
Experimental Procedures |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
Materials.
14C- Mannitol (molecular
weight, 182; specific radioactivity, 300 mCi/g) was obtained from New
England Nuclear Research Products (Boston, MA). C10 and C12 (99-100%
purity) and Flu (molecular weight, 376.3) were obtained from Sigma
Chemical (St. Louis, MO). Compound 48/80 (an inhibitor of PLC) (Bronner
et al., 1987), W7 (a calmodulin antagonist (Hidaka et
al., 1981; Itoh and Hidaka, 1984), ML7 (an inhibitor of MLCK)
(Saitoh et al., 1987) and H7 (an inhibitor of PKC) (Hidaka
et al., 1984) were obtained from Sigma Chemical. U73122 (an
inhibitor of PLC) (Bleasdale et al., 1990; Yule and
Williams, 1992), KN62 (an inhibitor of
Ca2+/calmodulin-dependent protein kinase II)
(Tansey et al., 1992) and calphostin C (an inhibitor of PKC)
(Kobayashi et al., 1989) were obtained from Calbiochem (San
Diego, CA). BAPTA-AM (a cell-permeant chelator of
Ca++) and diC8 (dioctanoylglycerol, a DAG analog)
were obtained from Molecular Probes (Eugene, OR). All tissue culture
media were obtained from GIBCO through Laboratorie Design AB
(Lidingö, Sweden).
Cells.
Caco-2 cells originating from a human colorectal
carcinoma (Fogh et al., 1977) were obtained from American
Type Culture Collection (Rockville, MD) and cultivated as described
previously (Anderberg et al., 1993; Artursson, 1990;
Artursson et al., 1996). In brief, 0.4 to 0.6 × 106 cells/cm2 were seeded
onto 12-mm-diameter polycarbonate filters (Transwell cell culture
inserts, mean pore diameter of 0.45 µm; Costar, Badhoevedorp, The
Netherlands). The cells were used for experiments 21 to 35 days after
seeding. Passages 90 through 106 were used.
Transport studies.
The transport of
14C-mannitol (trace amounts) or Flu (2 mg/ml),
with and without the addition of C10 or C12, across Caco-2 cell monolayers was studied as described previously (Artursson, 1990; Lindmark et al., 1995). All experiments were performed in
HBSS (containing 25 mM HEPES buffer, pH 7.4) at 37°C.
Ca++/Mg++-free HBSS was
always used in the donor chamber to avoid precipitation of C10 and C12.
Exclusion of Ca++/Mg++ from
the donor solutions did not affect the integrity of the monolayers
because the HBSS in the basolateral chamber always contained
Ca++/Mg++ (Anderberg
et al., 1993). The cell monolayers were equilibrated in
prewarmed HBSS for 20 min before the transport experiments. At
time = 0, the inserts were placed in new wells containing HBSS, and HBSS containing Flu or 14C-mannitol with or
without C10 or C12 was then added to the apical side of the cell
culture inserts. Samples were taken from the basolateral side by moving
the cell culture inserts to new basolateral chambers containing fresh
HBSS. The apparent permeability coefficient (Papp) was determined according to the following
equation: Papp = dQ/dt·1/ACo, where dQ/dt is the permeability
rate (steady-state flux; mol/sec), Co is the
initial concentration in the donor chamber (mol/ml) and A is the
surface area of the membrane (cm2).
Transmission electron microscopy. Monolayers were fixed with 1.5% glutaraldehyde, immersed consecutively in 1% osmium tetroxide and 1% uranyl acetate, dehydrated and embedded in Epon. Thin sections, stained with uranyl acetate and lead citrate, were examined with a Philips 420 electron microscope operated at 60 kV.
Intracellular enzyme activity (MTT).
Intracellular
dehydrogenase activity was determined using the MTT method (Mosman,
1983). Cells were incubated with serial dilutions of C10 or C12 in a
96-well plate for 60 min and assayed according to Anderberg et
al. (1992).
Measurement of ATP.
The ATP assay was based on the
luciferin/luciferase reaction, using an ATP detection kit (BioThema AB,
Dalarö, Sweden). This kit has a monitoring reagent formulated to
provide a time-independent signal (Lundin, 1990). After incubation, ATP
was extracted from the monolayers by the instantaneous addition of 1 ml
of 2.5% trichloroacetic acid, and ATP in the aliquot was measured.
Bioluminescence was measured with a 1250 Luminometer (LKB Wallac,
Turku, Finland). An internal standard was used to determine the ATP
concentration in each sample. ATP depletion, by exposing the cells to
antimycin A and 2-deoxyglucose (Bacallo et al., 1994), was
used as a positive control, decreasing the ATP levels to 2% of those
in control monolayers.
TER.
Caco-2 cells (grown on Snapwell cell culture inserts)
were mounted in Ussing-type chambers equipped with a four-electrode system; one pair of Pt electrodes was used for current passage, and one
pair of Ag/AgCl electrodes was used to measure the transepithelial potential difference. Measurements were made at 37°C in HBSS
(Karlsson, 1995). After mounting and equilibration in HBSS, the cell
monolayers were preincubated with 48/80 (2.5 µg/ml) for 20 min. Half
of the apical volume of HBSS was replaced with HBSS containing C10 or C12 (final concentrations in the apical chamber were 5 and 0.375 mM,
respectively) with or without 2.5 µg/ml 48/80; control cells contained HBSS only with 2.5 µg/ml 48/80. Measurements were made at
2- to 5-sec intervals the first 2 min and every minute thereafter. Average TER of the monolayers (n = 17) at time = 0 was 308.7 ± 59.2 
× cm2.
Immunohistochemistry and confocal microscopy. Before staining, the monolayers were exposed to buffer only (control), C10 (13 mM) or C12 (0.75 mM) for 12 or 60 min at 37°C. Further steps were performed at room temperature. Monolayers were fixed in 3% paraformaldehyde for 2 to 3 min and permeated with 0.2% Triton X-100 for 10 min. Rabbit polyclonal antibodies to ZO-1 (Zymed Lab, San Francisco, CA), diluted 1:1000 in PBS, or rabbit polyclonal antibodies against occludin (Zymed), final concentration of 10 µg/ml, were added to the apical side for 60 min. Excess solution was rinsed off, and the monolayers were incubated with PBS for 10 min. FITC-anti-rabbit antibody (Amersham, Buckinghamshire, England), diluted 1:100, was added to the apical side for 30 min. After a final incubation in PBS for 15 min, monolayers were mounted in Dako mounting medium (Dakopatts AB, Copenhagen, Denmark) on glass slides and examined under a Leica TCS 4D confocal laser scanning microscope equipped with a Leica Plan Apo 63× water-immersion lens with a numerical aperture of 1.20 and an argon/krypton laser, using the 488-nm line for excitation and a 530/30 emission filter. The images were processed with Image Space software (Molecular Dynamics, Sunnyvale, CA).
Statistics. All values are expressed as mean ± S.D. One-way analysis of variance (followed by the Fisher LSD for comparisons between two mean values) was used for the statistical analysis.
| |
Results |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
Short- and long-term effects on epithelial permeability. The MCFAs were studied at concentrations of 5 and 13 mM (C10) and 0.375 and 0.75 mM (C12), respectively. The lower concentrations (5 and 0.375 mM) were included to study more settled effects of the MCFAs. The two MCFAs had comparable time-dependent effects on epithelial permeability (fig. 1). Exposure to the lower C10 and C12 concentrations of 5 and 0.375 mM, respectively, revealed that both MCFAs rapidly and significantly enhanced the permeability of the monolayers within 2 to 4 min but that the epithelial integrity was restored after 20 to 40 min (fig. 1).
|
). After
a 2- to 3-min exposure of C10 or C12 (concentrations of 13 and 0.75 mM,
respectively), the permeability of the epithelium to Flu was
significantly increased (
3-fold) compared with control (fig. 1).
After this initial increase, the permeability remained at a constant
level for 20 min (C10) or 10 min (C12). At this time, a second increase
in permeability was initiated, which lasted throughout the 40-min
experiment, resulting in 7-fold increases in permeability on the
addition of C10 and C12 compared with control. Thus, the time-dependent
effects of C10 and C12 on epithelial permeability can be divided into
an initial phase lasting for 10 to 20 min, characterized by a rapid
increase in permeability, and a later phase, characterized by a slow
but more prolonged increase in permeability.
Effects on cellular metabolism. Routine transmission electron microscopy showed that some mitochondria in Caco-2 cells exposed to C10 (13 mM) for 60 min were swollen and rounded compared with cells exposed to C12 (0.75 mM) for 60 min or control cells (fig. 2), indicating changes in mitochondrial metabolism in the C10-treated cells. To further investigate this issue, the intracellular dehydrogenase activity and cellular ATP levels were investigated.
|
|
). Exposure to
13 mM C10, but not 0.75 mM C12, for 60 min resulted in a small but significant decrease in the ATP levels (fig.
3). Increasing the concentration of C10
to 16 mM and C12 to 1.0 mM further reduced the ATP levels. A shorter
exposure time (12 min), corresponding to that of the initial phase of
absorption enhancement in figure 1, did not decrease the ATP levels
(data not shown).
|
Inhibition of increase in epithelial permeability.
The effects
of a nonspecific inhibitor of PLC, 48/80, on the short-term effects of
C10 and C12 on epithelial permeability were investigated. C10 and C12
induced an instantaneous decrease in TER. Recovery in the direction of
base-line values after the initial decrease in TER occurred for both 5 mM C10 and 0.375 mM C12 in the presence of 48/80. Thus, recovery of
epithelial integrity was observed within 10 min (table
2). However, in contrast to a previous
observation (Tomita et al., 1995), 48/80 failed to inhibit
the effects of 13 mM C10 and 0.75 mM C12 (data not shown).
|
; Ghandehari et al., 1997; Hollander
et al., 1988; Lindmark et al., 1995).
|
|
Effects on tight junction-associated proteins.
Exposure to C10
(13 mM) or C12 (0.75 mM) for 12 min did not change the distribution of
the intracellular (ZO-1) and the extracellular (occludin) tight
junction proteins (data not shown). Exposure to C10, but not C12, for
60 min changed the appearance of both ZO-1 and occludin. In control
monolayers, continuous bands of ZO-1 and occludin were observed at the
cell borders (fig. 6, a and a
). After
exposure to C10 for 60 min, the ZO-1 and occludin staining became less
even and fragmented (fig. 6, b and b). However, all neighboring cells
remained in contact. C12 did not change the staining pattern of ZO-1 or
occludin compared with control (fig. 6c and c).
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
This report shows that C10 and C12, two absorption enhancers specifically acting on the tight junctions, regulate paracellular permeability through PLC-dependent IP3/DAG pathways. Surprisingly, the IP3 and DAG pathways were found to have opposing effects on paracellular permeability. Prolonged exposure to the enhancers also reduced cellular dehydrogenase activity and ATP levels, but this reduction was most likely not sufficient to mediate the increased permeability in the present study. These findings indicate that C10 and C12 have more complex mechanisms of action than previously recognized, which has several implications for their use as absorption enhancers.
The introduction of Flu as a sensitive paracellular marker molecule in
the studies of time-dependent absorption enhancement allowed the
identification of a previously unobserved rapid increase in
paracellular permeability. This rapid increase was followed by a second
phase characterized by a continued but slower increase comparable to
that observed previously after 20- to 60-min exposure to C10 and C12
(Anderberg et al., 1993; Lindmark et al., 1995). Although these results are in agreement with the rapid decrease in TER
in the present and a previous study (Anderberg et al., 1993), recent investigations have indicated that changes in TER and
mannitol permeability are not always correlated (Balda et al., 1996; Karlsson, 1995). It was therefore important to
establish that the rapid decrease in TER was reflected in a rapid
increase in marker permeability.
The increase in permeability was faster than that reported for other
tight junction selective absorption enhancers such as palmitoyl
carnitine and chitosans (Hochman et al., 1994; Schipper et al., 1996). A rapid onset of action is an advantageous
property in an absorption enhancer because after oral administration,
the intestinal epithelium is exposed to sufficiently high
concentrations of the absorption enhancer together with the drug for a
short time period only. Dilution in the luminal fluids and transport along the gastrointestinal tract will reduce the concentration of the
enhancer and drug at the epithelial surface. This does not mean that
the long-term effects are unimportant after local (e.g.,
rectal) administration because exposure of the mucosal tissue to the
enhancer is more sustained in this case (Lindmark et al.,
1997a).
Several reports suggest that a decrease in ATP levels may increase
paracellular permeability (e.g., Canfield et al.,
1991; Mandel et al., 1993). However, a 50% reduction in
the ATP levels was required to increase the permeability of rat ileal
mucosa in vivo (Madsen et al., 1995). Thus, it is
unlikely that the mechanism of absorption enhancement was related to
the modest changes in ATP levels observed in this study. Further
support for this hypothesis is provided by the fact that C12 did not
reduce the dehydrogenase or ATP levels although it increased the
permeability. A possible explanation for the effect of these MCFAs on
the ATP levels is that they may uncouple oxidative phosphorylation in a
way similar to that observed for another MCFA, octanoic acid (C8)
(Soboll et al., 1984).
Previous studies on the mechanism of C10 in Caco-2 monolayers suggested
that a pathway activated by PLC could be involved (Tomita et
al., 1995). The fact that the PLC inhibitor 48/80 aided recovery
of TER after short-term exposure to C10 or C12 in the present study
supports this hypothesis. In contrast to previous reports, no
inhibitory effect was observed of 48/80 on
14C-mannitol permeability (data not shown). A
possible explanation for this discrepancy is that a much larger marker
molecule was used in the previous study (Tomita et al.,
1995). A slight decrease in the tight junction pore size would reduce
the permeability of a large molecule, whereas it may not be sufficient
to reduce the permeability of a smaller marker molecule, such as
mannitol. However, because changes in mannitol permeability reflect
those of conventionally sized hydrophilic drugs (Anderberg et
al., 1993, Artursson and Karlsson, 1991; Lennernäs et
al., 1996), we retained mannitol as a marker throughout this
study. The finding that a more selective inhibitor of PLC, U73122,
inhibited the long-term effect of C10 on
14C-mannitol permeability gave further support
for the involvement of PLC in the mechanism of C10. Because there was
no corresponding inhibition of C12-induced absorption enhancement, the
role of PLC in the mechanism of action for this enhancer warrants
further investigation. The difference in the effect of U73122 on C10- and C12-mediated increase in permeability may be related to the higher
lipid solubility of C12, resulting in a more extensive accumulation of
C12 (compared with C10) in the cell membrane and a requirement of
higher concentration of U73122 to obtain inhibition.
Activated PLC cleaves PIP2 to the two
intracellular mediators IP3 and DAG.
IP3 releases Ca++ from
intracellular stores, thus increasing the intracellular Ca++ concentration, which could result in
increased paracellular permeability across the epithelium (Nathanson
et al., 1992; Tai et al., 1996). To investigate
whether elevated intracellular Ca++
concentrations are involved in the mechanisms of action of C10 and C12,
intracellular Ca++ ions were buffered with BAPTA.
This inhibited the effects of both C10 and C12, supporting previous
suggestions that elevated intracellular Ca++
levels is an important factor in the mechanism of C10 (Tomita et
al., 1995). The lack of effect of BAPTA on the short-term effect of C10 remains unexplained, but it is possible that use of a larger marker molecule or further titration of the BAPTA concentration could
provide evidence for the involvement of elevated
Ca++ concentration in this case (Tomita et
al., 1995).
It is well established that contraction of the cytoskeletal structure
adjacent to the tight junction and adherence junction results in
increased paracellular permeability (Madara et al., 1986,
1988). Contraction of this structure is driven by ATP-dependent interaction of myosin with the actin filaments that form the core structure of the terminal web (Citi and Kendrick-Jones, 1987; Keller
and Mooseker, 1982). The contraction is regulated by several mechanisms, including phosphorylation of the regulatory light chain of
myosin by Ca++/calmodulin-activated MLCK (Citi
and Kendrick-Jones, 1987; Keller and Mooseker, 1982). The finding that
an antagonist of calmodulin and a specific inhibitor of MLCK inhibited
the long-term effects of C10 and C12 on the permeability of the
epithelium to 14C-mannitol indicates that this
pathway is at least partly involved in mediation of the
absorption-enhancing effects of C10 and C12 in the intestinal
epithelium. Interestingly, inhibition of
Ca++/calmodulin kinase II, a multifunctional
Ca++/calmodulin-dependent kinase (Schulman, 1993;
Walsh, 1994), resulted in only inhibition of the effect of C10. Because
previous studies indicate that C10 (but not C12) induces morphological
changes in the perijunctional F-actin ring and in the tight junctions (Anderberg et al., 1993; Lindmark et al., 1995),
it may be speculated that Ca++/calmodulin kinase
II is involved in these changes. Because none of these inhibitors
affected the short-term increase in the permeability to
14C-mannitol caused by C10 and C12, we
tentatively conclude that the short-term effect of the MCFAs is
mediated via a partly different mechanism. This is supported
by results indicating that separations of the tight junctions induced
by C10 were first observed after long-term exposure to C10 (Anderberg
et al., 1993).
The second product of PLC-induced cleavage of
PIP2 is DAG. Along with
Ca++, DAG activates PKC, which in turn regulates
tight junction assembly as well as tight junction permeability (Balda
et al., 1991, 1993; Stenson et al., 1993; Stuart
and Nigam, 1995). The effects of PKC activation on tight junction
permeability are complex and vary with different experimental settings
and cell types (Ellis et al., 1992). Activation of PKC has
been shown to up- or down-regulate tight junction permeability
depending on the experimental environment (Balda et al.,
1993; Nathanson et al., 1992; Stenson et al.,
1993; Stuart and Nigam, 1995). The results of the
present study suggest that PKC activation down-regulated the
C10/C12-mediated effects on the paracellular permeability of Caco-2
cells. First, the application of PKC inhibitors at nontoxic
concentrations increased rather than decreased the C10- or C12-mediated
long-term effect on paracellular permeability. Second, the DAG agonist
diC8 inhibited the effects of the two MCFAs; this is in agreement with
studies on tight junction assembly in which diC8 was found to increase
the recruitment of the tight junction protein ZO-1 to the tight
junctions (Balda et al., 1993). We conclude that
PKC-mediated phosphorylation counteracts both the short- and long-term
effects of C10- and C12-mediated absorption enhancement in Caco-2
cells. Whether these effects are mediated via PKC isoenzymes
recently shown to be colocalized with ZO-1 at the tight junctions
remains to be seen (Dodane and Kachar, 1996; Stuart and Nigam, 1995).
The staining patterns of the tight junction proteins ZO-1 and occludin
after exposure to C10 suggest that these proteins are involved in the
morphological changes in tight junction structure after C10 exposure in
cell culture as well as in intestinal tissue (Anderberg et
al., 1993; Söderholm et al., 1995). Although no clear effects on the tight junctions were observed after C12 exposure, it cannot at present be excluded that changes below the resolution of
the confocal microscope occurred after exposure to C12.
The results of this study have several implications for the application
of C10 and C12 as absorption enhancers. First, the time- and
concentration-dependent effects on cellular ATP levels indicate that
prolonged interaction with an epithelial barrier (as obtained after
local administration, e.g., to the rectal mucosa; Lindmark
et al., 1997a) may result in nonspecific cell damage and
cell death caused by energy depletion. Development of delivery systems
that control the release of the absorption enhancer may solve this
problem. Second, the multiple and partly different intracellular
effects of C10 and C12 suggest that the efficacy and specificity of
these absorption enhancers may vary with the expression of, for
example, protein kinases and cytoskeletal components in different
epithelia (Ellis et al., 1992). Third, because many drug
molecules regulate intracellular events, such drugs could decrease or
increase the enhanced paracellular permeability. This could provide an
alternative explanation for the observed drug-dependent variability in
the efficacy of C10 in vivo (Aungst et al.,
1996).
| |
Acknowledgments |
|---|
We would like to thank Johan Gråsjö for assistance with the TER equipment, Dr. Göran Ocklind for making the confocal micrographs and Tapio Nikkilä for preparing the samples for electron microscopy.
| |
Footnotes |
|---|
Accepted for publication September 12, 1997.
Received for publication May 23, 1997.
1 This work was supported by grants from The Swedish Medical Research Council (9478), Centrala Försöksdjursnämnden (97-46), the Wallenberg Foundation and Astra AB.
2 Present address: Department of Food Science and Technology, Kyoto University, Japan.
3 Johan Gråsjö, unpublished observations.
Send reprint requests to: Prof. Per Artursson, Department of Pharmacy, Division of Pharmaceutics, Uppsala University, Box 580, S-75123 Uppsala, Sweden. E-mail: per.artursson{at}galenik.uu.se
| |
Abbreviations |
|---|
C10, sodium caprate;
C12, sodium laurate;
BAPTA-AM, the membrane-permeant acetoxymethyl ester of
1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid ;
DAG, diacylglycerol;
Flu, sodium fluorescein;
IP3, inositol
triphosphate, MCFA, medium chain fatty acid;
MLCK, myosin light chain
kinase;
PIP2, phosphatidylinositol bisphosphate, PKC,
protein kinase C;
PLC, phospholipase C;
TER, transepithelial electrical
resistance;
HBSS, Hanks' balanced salt solution;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium;
PBS, phosphate-buffered saline.
| |
References |
|---|
|
Top
Abstract
Introduction
Procedures
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Kondoh, A. Masuyama, A. Takahashi, N. Asano, H. Mizuguchi, N. Koizumi, M. Fujii, T. Hayakawa, Y. Horiguchi, and Y. Watanbe A Novel Strategy for the Enhancement of Drug Absorption Using a Claudin Modulator Mol. Pharmacol., March 1, 2005; 67(3): 749 - 756. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Suzuki and H. Hara Various Nondigestible Saccharides Open a Paracellular Calcium Transport Pathway with the Induction of Intracellular Calcium Signaling in Human Intestinal Caco-2 Cells J. Nutr., August 1, 2004; 134(8): 1935 - 1941. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Banan, L. J. Zhang, M. Shaikh, J. Z. Fields, A. Farhadi, and A. Keshavarzian Inhibition of Oxidant-Induced Nuclear Factor-{kappa}B Activation and Inhibitory-{kappa}B{alpha} Degradation and Instability of F-Actin Cytoskeletal Dynamics and Barrier Function by Epidermal Growth Factor: Key Role of Phospholipase-{gamma} Isoform J. Pharmacol. Exp. Ther., April 1, 2004; 309(1): 356 - 368. [Abstract] [Full Text]
|
|
|
|
|
|
S. Tavelin, K. Hashimoto, J. Malkinson, L. Lazorova, I. Toth, and P. Artursson A New Principle for Tight Junction Modulation Based on Occludin Peptides Mol. Pharmacol., December 1, 2003; 64(6): 1530 - 1540. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Banan, L. J. Zhang, M. Shaikh, J. Z. Fields, A. Farhadi, and A. Keshavarzian Key role of PLC-{gamma} in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly Am J Physiol Cell Physiol, October 1, 2003; 285(4): C977 - C993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. B. Coyne, C. M. P. Ribeiro, R. C. Boucher, and L. G. Johnson Acute Mechanism of Medium Chain Fatty Acid-Induced Enhancement of Airway Epithelial Permeability J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 440 - 450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. D. Ward, H. Ouyang, and D. R. Thakker Role of Phospholipase C-{beta} in the Modulation of Epithelial Tight Junction Permeability J. Pharmacol. Exp. Ther., February 1, 2003; 304(2): 689 - 698. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Banan, J. Z. Fields, D. A. Talmage, Y. Zhang, and A. Keshavarzian PKC-{beta}1 mediates EGF protection of microtubules and barrier of intestinal monolayers against oxidants Am J Physiol Gastrointest Liver Physiol, September 1, 2001; 281(3): G833 - G847. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Banan, J. Z. Fields, Y. Zhang, and A. Keshavarzian Phospholipase C-{gamma} inhibition prevents EGF protection of intestinal cytoskeleton and barrier against oxidants Am J Physiol Gastrointest Liver Physiol, August 1, 2001; 281(2): G412 - G423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Watson, M. Rowland, and G. Warhurst Functional modeling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomers Am J Physiol Cell Physiol, August 1, 2001; 281(2): C388 - C397. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H M Roche, A M Terres, I B Black, M J Gibney, and D Kelleher Fatty acids and epithelial permeability: effect of conjugated linoleic acid in Caco-2 cells Gut, June 1, 2001; 48(6): 797 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Banan, J. Z. Fields, Y. Zhang, and A. Keshavarzian Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants Am J Physiol Gastrointest Liver Physiol, May 1, 2001; 280(5): G828 - G843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. B. Coyne, M. M. Kelly, R. C. Boucher, and L. G. Johnson Enhanced Epithelial Gene Transfer by Modulation of Tight Junctions with Sodium Caprate Am. J. Respir. Cell Mol. Biol., November 1, 2000; 23(5): 602 - 609. [Abstract] [Full Text]
|
|
|
|
|
|
H. B Jijon, T. Churchill, D. Malfair, A. Wessler, L. D Jewell, H. G Parsons, and K. L Madsen Inhibition of poly(ADP-ribose) polymerase attenuates inflammation in a model of chronic colitis Am J Physiol Gastrointest Liver Physiol, September 1, 2000; 279(3): G641 - G651. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
), 5 mM (
) and 13 mM (
) C10. 5 mM C10 increased
Papp within 3 to 4 min (P < .05, vs. control). No further increase was observed at later time points; 13 mM
C10 increased Papp within 2 to 3 min of addition (P < .001). Papp was further increased at later time points
(P < .01). b, Control, no enhancer (
