Department of Physiology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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Expression of the Kir3
channel subfamily in gastrointestinal (GI) myocytes was investigated.
Members of this K+ channel subfamily encode G protein-gated
inwardly rectifying K+ channels
(IKACh) in other tissues, including
the heart and brain. In the GI tract, IKACh could
act as a negative feedback mechanism to temper the muscarinic response
mediated primarily through activation of nonselective cation currents
and inhibition of delayed-rectifier conductance. Kir3 channel subfamily
isoforms expressed in GI myocytes were determined by performing RT-PCR
on RNA isolated from canine colon, ileum, duodenum, and jejunum
circular myocytes. Qualitative PCR demonstrated the presence of Kir3.1
and Kir3.2 transcripts in all smooth muscle cell preparations examined.
Transcripts for Kir3.3 and Kir3.4 were not detected in the same
preparations. Semiquantitative PCR showed similar transcriptional
levels of Kir3.1 and Kir3.2 relative to -actin expression in the
various GI preparations. Full-length cDNAs for Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle RNA and coexpressed in Xenopus oocytes with human muscarinic type 2 receptor.
Superfusion of oocytes with ACh (10 µM) reversibly activated a
Ba2+-sensitive and inwardly rectifying K+
current. Immunohistochemistry using Kir3.1- and Kir3.2-specific antibodies demonstrated channel expression in circular and longitudinal smooth muscle cells. We conclude that an IKACh
current is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers.
colon; motility; smooth muscle; potassium; ion channel
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INTRODUCTION |
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ELECTRICAL RHYTHMICITY of gastrointestinal (GI) smooth
muscles modulates contractile activity of the muscle, leading to GI motility. The slow wave cycle ensures a period of relaxation between contractions to allow mixing and movement of luminal contents. Ionic
conductances expressed in interstitial cells of Cajal and smooth muscle
cells determine, to a large degree, the properties of the electrical
slow wave (6). Resting membrane potential in canine colonic smooth
muscle at the submucosal border is approximately 80 mV, close to
the K+ equilibrium potential (27). The Na+ pump
has been suggested to participate in the generation of the very
hyperpolarized resting potential in this tissue (2) but may not
play a significant role in the generation of pacemaking activity (1).
However, another factor that may influence resting potential in
these cells is K+ conductance in the form of strongly
inwardly rectifying K+ channels (Kir2.1) (5). This study
found that micromolar concentrations of Ba2+ inhibited slow
wave activity in the canine colon and depolarized resting membrane
potential. The identification of inwardly rectifying K+
conductance in colonic smooth muscle led us to question whether inwardly rectifying K+ channels might participate in the
response of the tissue to neurotransmitters.
ACh is an excitatory neurotransmitter in the gut (8). Application of 1 µM ACh to strips of colonic smooth muscle increases slow wave
duration and contractile activity (9). On the other hand, even under
this excitatory influence rhythmicity is not abolished and tonic
contraction does not occur. The primary targets of muscarinic
stimulation are nonselective cation channels (14). These are probably
activated through G protein -subunits and pass inward current
carried by Na+ and Ca2+, depolarizing membrane
potential and activating L-type Ca2+ channels. In addition
to this depolarizing current, muscarinic activation leads to the
inhibition of delayed-rectifier K+ channels in GI smooth
muscles (28), potentiating the excitatory response through a
prolongation of slow wave duration. Our hypothesis is that a
hyperpolarizing current may also be activated during muscarinic
stimulation to provide repolarization during excitatory stimulus,
maintaining rhythmicity and preventing a tonic contraction. Maintenance
of rhythmic contractile activity allows for increased motility under
excitatory conditions.
Members of the Kir3 family encode G protein-gated inwardly rectifying K+ channels (GIRKs) (4, 12). In cardiac myocytes, IKACh has been identified and characterized (3, 19, 23). Activation of this current slows heart rate and acts to hyperpolarize membrane potential. Krapivinsky et al. (11) identified the molecular components underlying this current to be a combination of Kir3.1 and Kir3.4. However, other combinations of Kir3 family members can encode a similar current (7, 18). IKACh could provide the hyperpolarizing current hypothesized to maintain rhythmic GI electrical and contractile activity during excitatory stimulus. We examined GI smooth muscles for the expression of Kir3 family members and demonstrated the presence of Kir3.1 and Kir3.2 but did not detect Kir3.3 or Kir3.4. We have cloned the GI smooth muscle forms of Kir3.1 and Kir3.2 and determined the properties of these channels expressed in oocytes. We conclude that these GIRKs encode an IKACh-like conductance in GI myocytes.
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MATERIALS AND METHODS |
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Isolation and collection of GI tissues and cells.
Mongrel dogs were overdosed with pentobarbital sodium (100 mg/kg), and
a midline incision was made along the abdomen. The stomach, small
bowel, and proximal colon were removed and immediately placed in Krebs
solution containing (in mM) 120.35 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 15.5 NaHCO3, 1.2 Na2HPO4, and 11.5 glucose (pH 7.4 after
equilibration with 95% O2-5% CO2 at
37°C). Segments of stomach, small bowel, and proximal colon were
pinned in a dissecting dish with the mucosa facing upwards, and the
overlying mucosa and submucosa were removed by sharp dissection. Strips
of circular smooth muscle (1 mm × 10 mm) were cut parallel to the
circular muscle axis (26). Smooth muscle cells from the circular muscle layer of the proximal colon, duodenum, ileum, and jejunum were enzymatically dispersed as previously described by Langton et al. (13).
Smooth muscle cells were transferred to the stage of a phase contrast
microscope and allowed to adhere to the glass coverslip bottom of the
chamber for 5 min. Smooth muscle cells were differentiated from other
cell types by their characteristic morphology: spindle-shaped cells
with a length of 50-100 µm and a width of 5-10 µm.
Through applied suction, single smooth muscle cells were collected by
aspirating them into a wide-bore patch-clamp pipette (borosilicate
glass; Sutter Instruments). Approximately 60 smooth muscle
cells were collected, flash-frozen in liquid nitrogen, and stored at
80°C until use.
RNA isolation and RT-PCR.
Total RNA was prepared from various tissues and smooth muscle cells by
use of the SNAP Total RNA Isolation kit (Invitrogen) per the
manufacturer's instructions. Polyinosinic acid (a carrier of RNA, 20 µg) was added to lysates because RNA was isolated from small amounts
of tissue (5-20 mg) or isolated smooth muscle cells (<60 cells).
First-strand cDNA was synthesized from RNA preparations using
Superscript II RNase H Reverse Transcriptase (GIBCO BRL). RNA (1 µg
for either quantitative or qualitative PCR studies performed on tissue;
1 pg for qualitative PCR on 60 single smooth muscle cells) was reversed
transcribed by use of an oligo(dT)12-18 primer (500 µg/µl). To perform PCR, the following sets of primers were used:
Kir3.1 sense nt 430-450 and antisense nt 751-770 (GenBank accession no. U39196); Kir3.2 sense nt 853-872 and
antisense nt 1161-1180 (L78480); Kir3.3 sense nt 960-980 and
antisense nt 1382-1402 (L77929); Kir3.4 sense nt 920-940 and
antisense nt 1193-1213 (L47208); and -actin sense nt
2383-2402 and antisense nt 3071-3091 (V01217). PCR primers
for
-actin were used to assess the viability of RNA samples as well
as to detect genomic DNA contamination, whereby the primers were
designed to span an intron in addition to two exons. In addition, c-Kit
primers (X06182, sense nt 2259-2283 and antisense nt
2873-2897) were used to detect interstitial cell contamination and
PGP9.5 primers (PGP9.5; D10699, sense nt 34-53 and antisense nt
344-363) were used to detect neuronal contamination. Complimentary
DNA (20% of the first-strand reaction) was combined with sense and
antisense primers (20 µM), 1 mM dNTPs, 40 mM
Tris · HCl (pH 8.3), 100 mM KCl, 3 units Taq
(Promega, Madison, WI), 1 Ampliwax Gem 100 (Perkin Elmer, Foster City,
CA), and RNase-free water to a final volume of 50 µl. PCR was
performed in a Perkin Elmer 2400 Thermal Cycler under the following
conditions: 32 cycles at 94°C for 15 s, 57°C for 20 s, 72°C
for 1 min, and then incubation at 72°C for 10 min. For
single-cell PCR, if no amplification product was detected in the first
round of amplification 10% of the first-round PCR products were added
to a new reaction mixture containing all of the components listed above
and 32 additional cycles of PCR were then performed. All PCR products
were separated by 2% agarose gel electrophoresis and sequenced by use
of an automated nucleotide sequencer (Applied Biosystems, model 310).
In every case throughout the study, amplification products of the
predicted size for the primer pairs were gel extracted and sequenced to
confirm their identity.
Quantitative PCR.
Quantitative PCR was performed by use of the PCR MIMIC construction kit
(Clontech), which is based on a competitive PCR approach; nonhomologous
engineered DNA standards (referred to as PCR MIMICs) compete with
target DNA for the same gene-specific primers. PCR MIMICs were
constructed for Kir3.1, Kir3.2, and -actin. Competitive PCR was
carried out by titration of sample cDNA with known amounts of the
desired nonhomologous PCR MIMIC constructs; 10-fold serial dilutions of
these constructs were then added to PCR amplification reactions. After
PCR, products were separated by 2% agarose gel electrophoresis and
quantified by use of Molecular Analyst.
Cloning and in vitro transcription of Kir3.1 and Kir3.2. Kir3.1 and Kir3.2 were cloned from colonic smooth muscle in the presence of gene-specific primers for Kir3.1 (sense nt 44-63 and antisense nt 1534-1553; D45022) and Kir3.2 (sense nt 474-498 and antisense nt 1936-1959; U11859), respectively. Full-length cDNA fragments were ligated into pCR2.1 vector constructs (Invitrogen) and transformed by use of the TA cloning kit (Invitrogen). Clones were then sequenced by use of an automated nucleotide sequencer (Applied Biosystems, Model 310). Kir3.1 and Kir3.2 capped RNA (cRNA) were transcribed in vitro from pCR2.1 plasmids containing full-length Kir3.1 or Kir3.2 cDNA by use of the Ambion mMessage mMachine transcription kit. Briefly, reactions contained 2-5 µg of linearized template DNA, 500 µM ribonucleotides, 1× transcription buffer, and 40 units T7 RNA polymerase; the final concentration of cRNA was adjusted to 1 ng/µl.
Oocyte isolation and injection. Adult female Xenopus laevis frogs (Xenopus Express, Homosassa, FL and Xenopus 1, Dexter, MI) were anesthetized in chilled 0.17% 3-aminobenzoic acid ethyl ester solution (Sigma). The ovarian lobes were removed and placed in ND96 solution plus 100 µg/ml gentamicin (Sigma). ND96 solution contained (in mM) 2.4 sodium pyruvate, 96 NaCl, 2 KCl, 1 MgCl2, 1.5 CaCl2, and 5 HEPES, pH 7.4. The lobes were mechanically opened and incubated in collagenase (type IA, 1.2 mg/ml; Sigma) in ND96 solution at room temperature for 2-3 h to remove the follicular layer. The oocytes were collected, rinsed, and stored in ND96 solution plus gentamicin (100 µg/ml) at 19°C for up to 24 h before injection. Stage V and VI oocytes were injected with 50 nl of mRNA encoding Kir3.1 and/or Kir3.2 plus the human M2 muscarinic receptor (hM2) to a total volume of 50 nl using a Drummond Nanoject microinjector (Drummond Scientific, Broomall, PA). The ratio of cRNA was adjusted so that the concentration of each cRNA was equal and a total volume of 50 nl was injected. The oocytes were then stored at 19°C for 2-5 days until electrophysiological assay.
Electrophysiological methods.
Whole cell K+ currents were recorded using the
two-microelectrode voltage-clamp technique (GeneClamp 500, Axon
Instruments, Foster City, CA). Microelectrodes were pulled from
glass capillaries (Kimas-51, Kimble Products) with resistances of
1-3 M when filled with 3 M potassium aspartate. Oocytes were
superfused with a low-Cl
, Ca2+-free
solution designed to minimize the endogenous Ca2+-activated
Cl
current in oocytes. This solution contained (in
mM) 90 potassium methanesulfonate, 2 MgCl2, 5 HEPES, and 0.05 niflumic acid, pH 7.4. In some cases, K+
was replaced by equimolar Na+. Reagents were
applied to the bath (volume 0.5 ml) via a gravity-fed perfusion system.
The dead time to exchange solutions was ~30 s. Each experiment was
performed at room temperature (24-28°C) on oocytes collected
from more than one frog. In some experiments, potassium
methanesulfonate was replaced with equimolar sodium methanesulfonate to
examine the K+ selectivity of the channel. Data were
collected using a GeneClamp 500 amplifier connected to a DigiData 1200 A/D converter (Axon Instruments) interfaced to a PC clone
microcomputer. Voltage protocols were applied using pCLAMP 6.0 software
(Axon Instruments). In short, 400-ms voltage steps were applied from a
holding potential of
10 mV to test potentials ranging from
100 mV to +50 mV in 10-mV increments. No correction for leak
subtraction was applied. Data were analyzed with Microcal Origin
software and expressed as means ± SE, with n representing the
number of oocytes. Statistical analysis was performed using the
Student's paired or unpaired t-test, and P values of
<0.05 were regarded as significant.
Solutions and drugs.
Stock solutions of ACh, atropine (1 mM; Sigma), and Ba2+ (1 M; Sigma) were prepared in distilled water. Immediately before use, stock solutions were diluted to the final desired concentration in
low-Cl, Ca2+-free solution.
Data analysis and statistical treatments
Qualitative PCR was performed on smooth muscle cells isolated from the
circular layer of the canine colon, duodenum, ileum, and jejunum from
at least three different dogs. For quantitative PCR studies, tissue
samples from two different animals contributed to each RNA preparation
and at least three different RNA isolations were performed. The
concentration of the target DNA as well as -actin was determined on
each sample; target DNA concentration was then normalized to
-actin
expression. All results are expressed as means ± SE, and n = number of experiments. Data for quantitative PCR were analyzed by
one-way ANOVA, and differences between tissues were illustrated by
Newman-Keuls multiple comparison tests. P values <0.05 were
considered significant.
Immunohistochemistry. Tissues from the proximal colon and jejunum were opened, and luminal contents were washed with Krebs-Ringer-bicarbonate solution. Tissues were pinned to the base of a Sylgard dish mucosal side up and fixed in 4% paraformaldehyde (wt/vol) made up in 0.01% PBS (0.1 M, pH 7.4) for 30 min at 4°C. After fixation, tissues were cut into slices longitudinally along the lumen and transversely across the lumen of the intestine using a scalpel. Tissues were then washed for 3 × 30 min in PBS. Tissues were cut into small muscle strips (2 × 10 mm) and cryoprotected in a graded series of sucrose solutions (5, 10, 15, and 20% wt/vol made up in PBS, 1 h each). Tissues were subsequently embedded overnight in a solution containing Tissue Tek (Miles) and 20% sucrose in PBS (1 part/2 parts vol/vol), and the following day they were rapidly frozen in isopentane precooled in liquid nitrogen. Cryosections were cut on a cryostat (Leica CM3050) at a thickness of 8 µm and collected on Vectabond-treated slides (Vector Laboratories, Burlingame, CA). Nonspecific antibody binding was reduced by incubation in 10% goat serum for 1 h at room temperature. Tissues were incubated overnight with either polyclonal anti-Kir3.1 (Alomone Labs) or polyclonal anti-Kir3.2 (Alomone Labs), both raised in rabbit at manufacturer's recommended dilutions. For negative control, primary antibody was omitted and PBS was added in its place. Immunoreactivity was detected using fluorescein FITC-conjugated secondary antibody (FITC anti-rabbit) at a dilution of 1:200 in PBS for 1 h at room temperature. Sections were then washed 3 × 15 min in PBS and mounted with an aqueous mounting medium (Aqua-Mount, Pittsburgh, PA). Mounted slides were viewed, and fluorescence photomicrographs were taken using a Nikon eclipse E600 fluorescence microscope with appropriate excitation and emission wavelengths for fluorescein FITC.
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RESULTS |
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Expression of Kir3 family members in GI smooth muscles.
The presence of the Kir3 channel subfamily in canine GI smooth muscle
cells was determined by performing RT-PCR on total RNA isolated from
proximal colon, duodenum, ileum, and jejunum circular smooth muscle
cell preparations. Qualitative RT-PCR was performed on myocytes that
were individually selected in an attempt to eliminate other
contaminating cell types from the analysis. Transcripts for Kir3.1 and
Kir3.2 (Fig. 1, A and B,
respectively) were observed in proximal colon, duodenum, ileum, and
jejunum smooth muscle cell preparations.
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Immunohistochemical localization of Kir3.1 and Kir3.2 in GI smooth
muscles.
Antibodies raised against Kir3.1 and Kir3.2 both exhibited positive
immunoreactivity within myocytes of both the jejunum of the small
intestine and the proximal colon of the large intestine. Positive
immunoreactivity was exhibited throughout the circular and longitudinal
muscle layers of the external muscularis (Fig. 3, A-D), within the muscularis
mucosa, and in a range of vasculature including both arteries and veins
(Fig. 3, A and D).
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Heterologous expression of Kir3.1 and Kir3.2 cloned from GI smooth
muscle.
Both Kir3.1 and Kir3.2 full coding sequences were cloned using RT-PCR
from RNA isolated from murine proximal colon smooth muscle (see
MATERIALS AND METHODS for details). The DNA sequences were
identical to those previously cloned from murine brain RNA. Xenopus oocytes were injected with cRNA encoding
hM2 alone, hM2 + Kir3.1, hM2 + Kir3.2, or hM2 + Kir3.1 and Kir3.2. Figure
4 shows representative currents elicited in
oocytes injected with cRNA encoding these Kir channels in control and
during exposure to 10 µM ACh, as well as the ACh-induced difference
current (i.e., the difference between control currents and currents in
the presence of ACh). Membrane currents in oocytes injected with
hM2 + Kir3.1 or hM2 + Kir3.2 were not
significantly different from those observed in oocytes injected with
hM2 alone. In contrast, injection with cRNA encoding
hM2 + Kir3.1 and Kir3.2 resulted in the heterologous expression of inwardly rectifying currents (Fig. 4 and Fig.
5C). These data suggest that Kir3.1
and Kir3.2 have, at best, limited ability to form functional channels
on their own and that coexpression results in the expression of
significantly larger current (17). We also observed that inward
currents in the hM2 + Kir3.1 and Kir3.2 oocytes peaked
later than the inward currents in the other oocytes, suggesting that
the kinetics of the endogenous currents differ from those of the
heterologously expressed inwardly rectifying currents.
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DISCUSSION |
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We have shown that the IKACh channels Kir3.1 and Kir3.2 are expressed in GI smooth muscles as well as myocytes using a combination of RT-PCR on RNA from isolated cells, quantitative RT-PCR on RNA from bulk tissue, and immunohistochemistry using commercially available specific antibodies. Other known members of the Kir3 family (e.g., Kir3.3 and Kir3.4) have not been detected in these cells. This is the first report of Kir3 expression in smooth muscles. When expressed heterologously, the smooth muscle forms of Kir3.1 and Kir3.2 are inwardly rectifying K+ channels that are activated through muscarinic receptors. When they are expressed individually, very little current is displayed. Only when they are expressed together, presumably as a heterotetramer (11), are large inwardly rectifying currents produced. In addition, currents are observed when Kir3.1 and Kir3.2 are expressed without coexpression of the muscarinic receptor hM2 but are unaffected by ACh application. When coexpressing hM2 with Kir3.1 and Kir3.2, a large basal current is observed that can be potentiated by application of ACh. The properties of this heterotetrameric channel differed only slightly from a previous report (17). The Ba2+ sensitivity of our clones is higher (44 µM vs. 105 µM).
Muscarinic stimulation of GI muscles leads to an increased force of
contraction and an increased rate of GI motility (24, 25). The
mechanism proposed for mediating the increased excitability involves
neurotransmitter binding to M2 and M3 receptors
on the smooth muscle cell surface (30, 31). M2 receptors
are coupled to the Gi/Go family of G proteins,
whereas M3 receptors are coupled to pertussis
toxin-insensitive Gq/11 that activates
membrane-bound phospholipase C- (PLC-
). M2 receptor
stimulation leads to the inhibition of adenylyl cyclase and protein
kinase A (PKA), which would have an excitatory effect due to the
stimulatory influence of PKA on several K+ channels in GI
muscles (10). M3 receptor stimulation coupling through
PLC-
and Gq would have at least two targets
that both eventually lead to the activation of L-type Ca2+
channels and release of Ca2+ from internal stores.
Nonselective cation currents are activated by excitatory
neurotransmitters, such as ACh, that result in depolarization and
activation of L-type Ca2+ channels (14, 16). In addition,
PLC-
stimulation leads to protein kinase C activation that inhibits
delayed-rectifier currents in GI smooth muscles, also contributing an
excitatory influence (28). The concomitant activation of
IKACh (Kir3.1/3.2) in these muscles would temper
the excitatory response and prevent the tonic contraction that would
inhibit motility. Unlike tonic smooth muscles, phasic muscles require
rhythmic contractions to maintain functional activity. With the
inhibition of delayed-rectifier K+ channels as a result of
muscarinic activation, IKACh may provide the
hyperpolarization necessary for rhythmic oscillations in membrane potential. Activation of IKACh is proposed to be
through direct G protein
-stimulation (29) that in GI muscles
could result from stimulation of M2 or M3.
We observed basal activity of Kir3.1/3.2 when expressed with the
M2 muscarinic receptor. These results are consistent with those of other investigators (12, 29). The cardiac muscarinic K+ channel is not active in the absence of either receptor
stimulation or direct G protein -subunit application (19).
Neuronal G protein-gated inwardly rectifying K+ channels
are also inactive under basal conditions and can be isolated from the
more Ba2+-sensitive and strongly rectifying channels of the
Kir2 family (21, 22). In GI smooth muscle cells, the effects of
muscarinic stimulation on K+ currents at very
hyperpolarized potentials have yet to be examined. In addition, the
possibility of a basally active IKACh-like
conductance contributing to resting membrane potential along with
Kir2.1 currents (5) is difficult to assess because of the lack of
appropriate pharmacological tools. However, the molecular
identification of the components for an IKACh-like
current suggests a role for this conductance during excitatory
neurotransmission. The results from this molecular study should
stimulate interest in IKACh as a component of the
muscarinic response in GI smooth muscles and lead to investigations of
the regulation of this conductance in native myocytes.
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ACKNOWLEDGEMENTS |
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We thank Felicitas Griffin and Lisa Miller for technical assistance.
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FOOTNOTES |
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National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant DK-41315 supported this work.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. Horowitz, Dept. of Physiology, Univ. of Nevada School of Medicine, Reno, NV 89557 (E-mail: burt{at}physio.unr.edu).
Received 6 July 1999; accepted in final form 25 October 1999.
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