Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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We investigated the regulation of
ATP-sensitive K+ (KATP) currents in murine
colonic myocytes with patch-clamp techniques. Pinacidil
(105 M) activated inward currents in the presence of high
external K+ (90 mM) at a holding potential of
80 mV in
dialyzed cells. Glibenclamide (10
5 M) suppressed
pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 10
7 M) inhibited pinacidil-activated current.
4-
-Phorbol ester (5 × 10
7 M), an inactive form
of PDBu, had no effect on pinacidil-activated current. In cell-attached
patches, the open probability of KATP channels was
increased by pinacidil, and PDBu suppressed openings of
KATP channels. When cells were pretreated with
chelerythrine (10
6 M) or calphostin C (10
7
M), inhibition of the pinacidil-activated whole cell currents by PDBu
was significantly reduced. In cells studied with the perforated patch
technique, PDBu also inhibited pinacidil-activated current, and this
inhibition was reduced by chelerythrine (10
6 M).
Acetylcholine (ACh; 10
5 M) inhibited pinacidil-activated
currents, and preincubation of cells with calphostin C
(10
7 M) decreased the effect of ACh. Cells dialyzed with
protein kinase C
-isoform (PKC
) antibody had normal responses to
pinacidil, but the effects of PDBu and ACh on KATP were
blocked in these cells. Immunofluorescence and Western blots showed
expression of PKC
in intact muscles and isolated smooth muscle cells
of the murine proximal colon. These data suggest that PKC regulates KATP in colonic muscle cells and that the effects of ACh on
KATP are largely mediated by PKC. PKC
appears to be the
major isozyme that regulates KATP in murine colonic myocytes.
potassium channel openers; glibenclamide; protein kinase C
inhibitors; protein kinase C -isoform
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INTRODUCTION |
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SINCE THE IDENTIFICATION OF a K+ current in cardiac myocytes that was inhibited by intracellular ATP (24), there have been many reports that ATP-sensitive K+ (KATP) channels play important roles in regulating resting membrane potential and membrane excitability in a variety of tissues. The common properties of KATP channels are that they are 1) activated by K+ channel agonists such as lemakalim, diazoxide, and pinacidil, 2) inhibited by glibenclamide, and 3) modulated by intracellular ATP and nucleotide diphosphates (3, 6, 13, 30).
In previous studies of murine colonic muscle cells, KATP channels were found to be activated under resting conditions (17). Application of glibenclamide depolarized membrane potential and increased excitability. K+ channel openers, such as lemakalim and pinacidil, hyperpolarized intact muscles and blocked the spontaneous discharge of action potentials. The single-channel conductance activated by K+ channel openers was 27 pS in symmetrical K+ gradients. Molecular studies showed the expression of the ionic channel of KATP (Kir 6.2) and the sulfonylurea receptor type 2B (SUR2B) in murine colonic myocytes. Intracellular ATP regulates the open probability of KATP channels, but ATP is normally in the millimolar range in smooth muscle myocytes (1, 26). Nucleotide diphosphate also contributes to the regulation of open probability (13). For example, we found that after rundown of KATP channels in patches excised from colonic myocytes, either high concentrations of ADP (1 mM) or low concentrations of ATP (0.1 mM) applied to the intracellular surface could restore openings of KATP channels. Therefore, the ratio of ADP to ATP may be an important factor regulating these channels. Agonists and second messengers may also regulate KATP channels in colonic myocytes.
In vascular and visceral smooth muscle, vasodilating substances such as
adenosine, isoproterenol, and calcitonin gene-related peptide activate
KATP channels via cAMP-dependent mechanisms (22, 23). On the other hand, excitatory agonists such as angiotensin II and carbachol inhibit KATP channels in vascular,
tracheal, bladder and esophageal smooth muscles via protein kinase C
(PKC)-dependent mechanisms (1, 6a, 7, 18, 25). In the present study, we
have characterized the regulation of KATP channels in
gastrointestinal muscle cells by PKC -isoform (PKC
), which has
not been previously investigated. These studies were performed on
isolated murine colonic myocytes by using patch-clamp techniques.
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METHODS |
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Cell isolation. Colonic smooth muscle cells were isolated from 20- to 30-day-old BALB/C mice of either sex. Mice were anesthetized with chloroform and killed by cervical dislocation, and the proximal colon was quickly removed. The colon was opened along the myenteric border, and mucosa and submucosa were removed in Ca2+-free Hanks' solution containing (in mM) 125 NaCl, 5.36 KCl, 15.5 NaOH, 0.336 Na2HCO3, 0.44 KH2PO4, 10 glucose, 2.9 sucrose, and 11 HEPES. Strips of colonic muscle were transferred to the same buffer solution containing 230 units of collagenase (Worthington Biochemical), 2 mg of fatty acid-free bovine serum albumin (Sigma Chemical), 2 mg of trypsin inhibitor (Sigma Chemical), and 0.1 mg of papain (Sigma Chemical). Incubation in the enzyme solution was carried out at 37°C for ~10-12 min, and then the tissues were washed with Ca2+-free Hanks' solution. Single cells were obtained by gentle agitation with a wide-bored glass pipette. Isolated cells were kept at 4°C until they were used. Before electrophysiological studies were begun, a drop of the cell suspension was pipetted into a small chamber (0.3 ml) on the stage of an inverted microscope. The experiments were carried out within 6 h of dispersing the cells. All electrophysiological recordings were performed at room temperature (22-25°C).
Voltage-clamp experiment.
Patch-clamp experiments using the dialyzed whole cell, perforated whole
cell, and cell-attached patch configurations were performed on colonic
smooth muscle cells. Glass pipettes with resistance of ~3-5 M
for whole cell configuration and 5-10 M
for cell-attached
single-channel recordings were used. Membrane currents were amplified
by an Axopatch 1-D (Axon instruments) or an Axopatch 200B amplifier and
CV-4 head stage (Axon Instruments, Foster City, CA). Command pulses
were applied with an IBM-compatible personal computer and pCLAMP
software (version 5.5 or 6.1; Axon instruments). The data were filtered
at 5 kHz and displayed on an oscilloscope, a computer monitor, and a
pen recorder (Gould, Valley View, OH). To minimize activities of
voltage-dependent K+ channels and
Ca2+-activated K+ channels, we performed the
whole cell experiments at a holding potential of
80 mV in a solution
containing elevated extracellular K+ concentration (see below).
Solutions and drugs. For dialyzed whole cell recordings, the internal pipette solution contained (in mM) 10 NaCl, 102 KCl, 1 CaCl2, 1 GTP, 10 HEPES, 10 EGTA, 0.1 ATP, and 1 MgCl2, adjusted to pH 7.2 with KOH (38 mM). The normal external solution contained (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, and 0.2 CaCl2, adjusted to pH 7.4 with Tris. The high-K+ external solution contained K+ (90 mM) and Na+ (50 mM). For amphotericin B perforated patches, the composition of pipette solution (in mM) was 110 K-gluconate, 30 KCl, 5 MgCl2, and 5 HEPES, adjusted to pH 7.2 with Tris. Final concentration of amphotericin B was 270 µg/ml.
For cell-attached patch recordings, the external solution contained (in mM) 140 KCl, 1 EGTA, 0.2 CaCl2, and 10 HEPES, adjusted to pH 7.4 with Tris. The pipette solution contained (in mM) 135 NaCl, 5 KCl, 1 MgCl2, and 10 HEPES, adjusted to pH 7.4 with Tris. Charybdotoxin (200 nM) was included in the pipette solution in the majority of cell-attached patch experiments to inhibit large-conductance Ca2+-activated K+ channels. Pinacidil, calphostin C, and chelerythrine were purchased from RBI. Glibenclamide, 4-Immunohistochemistry.
The proximal colon segments were opened along the mesenteric border,
and luminal contents were washed away with Krebs Ringer bicarbonate
solution (KRB). The opened segments were pinned onto the base of a
Sylgard dish with the mucosal side facing up. The mucosa was removed by
sharp dissection, and the remaining tunica muscularis was fixed with
4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min at
4°C. After fixation, preparations were washed for 30 min in
phosphate-buffered saline (PBS; 0.05 M, pH 7.4). Nonspecific antibody
binding was reduced by incubation of the tissues in 10% normal goat
serum for 1 h at room temperature. Tissues were incubated with the
primary antibody (polyclonal rabbit antiserum against PKC; 1:150;
Boehringer Mannheim) for 48 h at 4°C. The tissues were then
exposed to FITC-coupled goat anti-rabbit IgG (1:100; Vector
Laboratories, Burlingame, CA) for 1 h at room temperature. Control
tissues were prepared by omitting either primary or secondary
antibodies from incubation solutions. All the antisera were diluted
with 0.3% Triton X-100 in 0.05 M PBS (pH 7.4). Tissues were examined
with a Bio-Rad MRC 600 confocal microscope (Hercules, CA) with an
excitation wavelength appropriate for FITC (494 nm).
Indirect immunofluorescence of PKC in isolated smooth muscle
cells.
Murine proximal colon smooth muscle cells were enzymatically dispersed
and incubated in physiological solution. After attachment to glass
coverslips for 2-3 h, the cells were rinsed twice with PEM (100 mM
PIPES, pH 7.4, 2 mM EGTA, and 1 mM Mg-acetate) and then incubated in
PEM/3.7% formaldehyde for 30 min. The PEM/3.7% formaldehyde was
gently aspirated, and the cells were rinsed twice with PBS and then
incubated for 5 min in PBS/100 mM glycine to quench any remaining
formaldehyde. The cells were then extracted in PEM/0.3% Nonidet P-40
for 10 min. The detergent extraction was stopped by two rinses with
PBS, and the cells were stored in PBS at 4°C before indirect
immunofluorescence staining.
Western blotting.
Colon smooth muscle tissues were obtained as described in
Immunohistochemistry. Smooth muscle lysates were
obtained by ground glass homogenization in 40 mM Tris · HCl, pH
7.5, 6 mM MgSO4, 1 mM EGTA, 0.1% Triton X-100, 0.5 mM
dithiothreitol, and protease inhibitors. The crude homogenate was
centrifuged at 10,000 g at 4°C for 15 min. The supernatant
was analyzed for protein content by using the Bradford assay, with
bovine gamma globulin as standard. The supernatant was separated by
SDS-PAGE (10%) and electroblotted onto nitrocellulose. The blots were
incubated in blocking buffer (PBS, 0.2% casein, and 0.1% Tween 20)
for 45 min, followed by incubation with rabbit anti-PKC antibody
(1:200 dilution; PanVera) in blocking buffer for 90 min. To neutralize
the antibody, we incubated a 10-fold molar excess of antigenic peptide
with the antibody for 1 h before the blots were incubated. The
blots were washed with two 5-min washes in blocking buffer minus
casein, followed by incubation with alkaline phosphatase-conjugated
goat anti-rabbit IgG antibody (1:5,000 dilution; Tropix) in blocking buffer for 90 min. The blots were washed as before and then washed with
two 2-min washes in assay buffer (20 mM Tris · HCl, pH 9.8, and
1 mM MgCl2). Immunodetection was carried out by using the Western-Star enhanced chemiluminescence kit (Tropix).
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RESULTS |
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To avoid contamination from voltage-dependent currents expressed
by murine colonic myocytes, cells were held at 80 mV, and the
external bathing solution was changed from 5 to 90 mM K+.
Activation of a K+ conductance under these conditions
results in inward current. Switching from 5 to 90 mM external
K+ resulted in a steady-state inward current that averaged
58.2 ± 8.4 pA (n = 14 experiments).
Pinacidil (10
5 M) activated additional inward currents
with a mean amplitude of
429 ± 13 pA (n = 14),
and glibenclamide (10
5 M) suppressed 90 ± 3% of
the pinacidil-activated currents (n = 7, Fig.
1A). PDBu (2 × 10
7 M), an activator of PKC, inhibited
pinacidil-activated currents by 67 ± 6% (n = 8)
slowly in dialysis patch, and the remaining currents were returned to
baseline by 5 mM K+-containing solution (Fig.
1B). On the other hand, 4-
-phorbol (5 × 10
7 M), an inactive form of PDBu, had no effect on the
pinacidil-activated currents in four cells (Fig. 1C).
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We reported the single-channel conductance of KATP in
murine colonic myocytes previously (i.e., 27 pS in symmetrical 140/140 mM K+) (17). In the present study the open
probability of KATP channels was measured in cell-attached
patches. Pinacidil activated K+ channels at 0 mV in 5 mM
extracellular/140 mM intracellular K+ concentrations (Fig.
2, A and B). The
control open probability was 0.10 ± 0.05 (Fig. 2D).
After treatment with pinacidil, open probability increased to 0.38 ± 0.10 (Fig. 2E). Addition of PDBu (5 × 107 M) to the same cells suppressed the openings of
KATP channels (0.08 ± 0.09; n = 3)
(Fig. 2, C and F).
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We tested whether activation of PKC was responsible for the inhibitory
effects of PDBu on KATP by pretreating cells with PKC inhibitors (Fig. 3). Inhibitors with
different mechanisms of action were chosen. Chelerythrine is a potent
PKC inhibitor that binds in a noncompetitive manner to the ATP binding
site (8). Calphostin C inhibits PKC by binding to the
phorbol ester binding site (4). Pretreatment with either
chelerythrine (106 M) (Fig. 3A) and calphostin
C (10
7 M) (Fig. 3B) for 20 min had no effect
on the activation of current by pinacidil, but these compounds reduced
the effects of PDBu on the current [i.e., to 17 ± 5%
(n = 5) and 14 ± 4% (n = 5)], respectively (Fig. 3C).
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We also performed experiments by using the perforated patch
technique to test the effects of PDBu on the pinacidil-activated current in nondialzyed cells. The amplitude of pinacidil-activated currents was 288 ± 20 pA (n = 4). PDBu
inhibited pinacidil-activated currents by 92 ± 4%
(n = 5) in cells studied with the perforated patch
technique (Fig. 4, A and
C). This was a greater degree of inhibition than observed in
dialyzed cells, suggesting that diluting the soluble components of the
cytoplasm with the pipette solution reduced the effectiveness of PKC
regulation. Chelerythrine (10
6 M) reduced the inhibition
of the pinacidil-activated current by PDBu to 16 ± 5%
(n = 5) (Fig. 4, B and C).
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Acetylcholine (ACh), a primary excitatory neurotransmitter in the
gastrointestinal (GI) tract, has been shown to activate PKC in some
smooth muscles (28). Therefore, we exposed dialyzed cells
to ACh (105 M) and found that this treatment reduced the
pinacidil-activated current by 58 ± 5% (n = 5)
(Fig. 5, A and C).
Preincubation of the cells with calphostin C (10
7 M)
decreased the inhibitory effects of ACh (to 10 ± 5%,
n = 5) (Fig. 5, B and C).
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PKC has been implicated in mediating responses to several agonists,
including ACh, in smooth muscles (9, 28). Therefore, we
performed experiments in which cells were dialyzed with an antibody
raised against a PKC
-specific epitope to test whether this isozyme
participates in regulation of KATP. After dialysis with
anti-PKC
, PDBu inhibited the pinacidil-activated current by only
12 ± 3% (n = 5) (Fig.
6, A and D). When
cells were dialyzed with anti-PKC
antibody that had been heat
inactivated, PDBu inhibited the pinacidil-activated current by 59 ± 7% (n = 5) (Fig. 5, B and D),
which was not significantly different from the effect of PDBu in
control studies. Similarly, when cells were dialyzed with PKC
antibody, ACh had only a small effect on KATP current (reduced ACh inhibition to 10 ± 5%, n = 5) (Fig.
6, C and D).
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Immunohistochemical experiments were also performed to determine
whether murine colonic muscle cells express PKC immunoreactivity. With the Boehringer Mannheim anti-PKC
antibody, prominent
immunoreactivity was observed in smooth muscle cells and enteric
neurons (Fig. 7). Circular and
longitudinal muscle cells were immunopositive (Fig. 7A). In
testing the specificity of the Boehringer Mannheim anti-PKC
antibody
(i.e., Western blots of purified PKC isozymes; data not shown), it was
recognized that the Boehringer Mannheim anti-PKC
antibody
demonstrated cross-reactivity with PKC
. Therefore, we tested
immuoreactivity of smooth muscle cells by using a second anti-PKC
antibody (PanVera; highly selective for PKC
) and found isolated
colonic myocytes to be immunopositive (Fig. 7, B and C). To further investigate the expression of PKC
in
murine proximal colon smooth muscle, we carried out Western blot
analysis of colonic smooth muscle lysates using the PanVera anti-PKC
antibody. As shown in the Western blot in Fig.
8, the purified PKC
migrates in
SDS-PAGE to its expected molecular mass of ~90 kDa. In addition, a
single immunoreactive band between 75 and 100 kDa that comigrates with
purified PKC
in SDS-PAGE was detected in proximal colon smooth
muscle lysates. The immunodetection of this band was blocked by
preincubation of the anti-PKC
antibody with the antigenic peptide
(Fig. 8, lane 3).
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DISCUSSION |
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In a previous study we found that KATP contributes to the regulation of membrane potential and excitability in murine colonic muscles (17). Glibenclamide blocked resting KATP conductance, produced depolarization, and increased action potential generation. Lemakalim and pinacidil, which are activators of KATP, increased the open probability of single KATP channels, hyperpolarized muscles, and inhibited spontaneous action potentials. Activation of KATP channels under basal conditions was also observed in pig proximal urethra (31). Thus modulation of the open probability of KATP channels could provide a means of regulating excitability in some smooth muscles. The fact that KATP contributes to the resting conductance of smooth muscle cells suggests that inhibition of this current could be a mechanism by which excitatory transmitters or hormones could act.
Regulation of KATP channels in smooth muscles occurs by
several mechanisms. Cytoplasmic ATP levels are usually at millimolar levels (1, 26) and fall substantially only under
conditions of severe metabolic inhibition. Cytoplasmic ATP may
therefore set a relatively low open probability for the channels under
basal conditions, against which other regulator factors may modulate channel openings (27). Studies have also shown that the
open probability of KATP in smooth muscles is regulated by
nucleotide diphosphate concentrations (13), and this
property of the isoform(s) of KATP expressed by smooth
muscle may enhance open probability over that predicted from the high
levels of ATP. KATP channels are also known to be activated
by a variety of vasodilators through protein kinase A (23, 26,
27). In contrast, several vasoconstrictors are coupled through G
proteins to phospholipases, generation of inositol 1,4,5-trisphosphate
and diacylglycerol, and activation of PKC. Previous studies of
vascular, tracheal, and bladder smooth muscle cells have shown that
stimulation by excitatory agonists inhibits KATP via
activation of PKC (1, 2, 6a, 18, 25). The present study sought to
determine whether PKC regulates KATP in GI smooth muscle
cells and whether this mechanism might mediate some of the excitatory
effects of cholinergic stimulation. In rabbit esophageal smooth muscle,
KATP was suppressed by phorbol 12-myristate 13-acetate, and
these effects were significantly reduced by PKC inhibitors (7). Our results have extended previous studies by showing that a specific isoform of PKC (PKC) is expressed in colonic muscles
and appears to be largely responsible for regulation of KATP.
PKC comprises one of the major second messenger systems that mediate
the responses to agonists in smooth muscles. There are several isozymes
of PKC, some of which are translocated from the cytosol to the cell
membrane upon stimulation (14, 15). PKC isozymes can be
activated by diacylglycerol, intracellular Ca2+, or
phospholipids under physiological conditions (29).
PKC and PKC
undergo agonist-induced translocation from the
cytosol to the plasma membrane in vascular smooth muscle cells
(15, 20, 28). Activation and translocation of the
Ca2+-dependent isozyme, PKC
, in vascular smooth muscle
cells occurs at basal or near basal levels of intracellular
Ca2+ concentration (15). In contrast PKC
,
which has been implicated in contraction of ferret aorta smooth muscle
cells, translocates from the cytosol to the surface membrane under
low-Ca2+ conditions and may mediate
Ca2+-independent contractions in these cells (9, 14,
16, 33). PKC
also contributes to the mediation of agonist
responses in GI muscles (28); however, the full range of
substrates for PKC
in GI muscle cells has not been described. In the
present study we have provided data showing that Kir 6.2 (17), SUR2B (17), or a subsidiary regulatory
protein may be a substrate for PKC
in colonic muscle cells. Dialysis
of cells with a specific antibody against PKC
blocked most of the
inhibitory effects of PDBu on KATP. In addition,
immunohistochemical and Western analyses demonstrate the presence of
PKC
in murine proximal colon smooth muscles.
The inhibitory effects of PKC on KATP open probability may be related to the specific isoforms of Kir 6 or sulfonylurea receptors (SUR) expressed in smooth muscles. For example, in rabbit and human ventricular myocytes, which express Kir 6.2 and SUR2A (18, 19), PKC activated KATP by reducing channel sensitivity to intracellular ATP (10). In contrast, in epithelial cells of the renal proximal tubule, KATP channels were inhibited by phorbol ester in cell-attached patches, and PKC applied to excised patches decreased open probability (21). Similar regulation was observed in the present study in cells that express Kir 6.2 and SUR2B (17). Because the primary difference between colonic KATP and cardiac KATP appears to be the isoform of SUR associated with the channels, it may be that the primary regulation by PKC occurs via this subunit.
The present study provides a new mechanism by which cholinergic stimulation might enhance the excitability of GI smooth muscles. ACh stimulates nonselective cation channels in GI muscles (5, 11, 12, 32), and this effect would be expected to result in depolarization and an increase in excitability. Inhibition of K+ channels that are open under basal conditions could also provide excitatory input in these cells. Many of the K+ channels expressed by GI muscle cells are activated by depolarization and have low open probabilities at resting potentials. Previous studies have demonstrated basal activation of KATP (17), so suppression of these channels would have a depolarizing influence on colonic cells. Together with the nonselective cation conductance expressed by many GI muscles and activated by excitatory neurotransmitters, inhibition of KATP by PKC may be an important excitatory mechanism.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mike Walsh (Univ. of Calgary) for helpful discussions.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41315 and DK-57168 (to B. Perrino). J. Y. Jun was supported by Chosun University (Republic of Korea), the Korean Ministry of Science and Technology, and the Korean Science and Engineering Foundation through the Research Center for Proteinous Materials.
Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (E-mail: kent{at}physio.unr.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 12 November 2000; accepted in final form 24 April 2001.
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