Distribution of heme oxygenase and effects of exogenous carbon
monoxide in canine jejunum
G.
Farrugia1,2,
S. M.
Miller1,
A.
Rich1,
X.
Liu1,
M. D.
Maines3,
J. L.
Rae1, and
J. H.
Szurszewski1
1 Department of Physiology and
Biophysics and 2 Division of
Gastroenterology and Internal Medicine, Mayo Clinic and Mayo
Foundation, Rochester, Minnesota 55905; and
3 Department of Biophysics, University of Rochester
School of Medicine, Rochester, New York 14642
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ABSTRACT |
Carbon monoxide (CO) has been postulated to be a
messenger in the gastrointestinal tract. The aims of this study were to
determine the distribution of heme oxygenase (HO), the source for
endogenous CO in the canine jejunum, and to determine the effects of CO
on jejunal circular smooth muscle cells. HO-2 isoform was present in a
population of myenteric and submucosal neuronal cell bodies, in nerve
fibers innervating the muscle layers, and in smooth muscle cells. HO-1
isozyme was not detected in the canine jejunum. Exogenous CO increased
whole cell current by 285 ± 86%, hyperpolarized the membrane
potential by 8.5 ± 2.9 mV, and increased guanosine
3',5'-cyclic monophosphate (cGMP) levels in
smooth muscle cells. 8-Bromo- cGMP also increased the whole
cell current. The data suggest that endogenous activity of HO-2 may be
a source of CO in the canine jejunum and that exogenously applied CO
can modulate intestinal smooth muscle electrical activity. It is
therefore reasonable to suggest a role for endogenously produced CO as
a messenger in the canine jejunum.
potassium channels; smooth muscle
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INTRODUCTION |
CARBON MONOXIDE (CO) is a low molecular weight gas that
is produced under physiological conditions (see Ref. 11 for review). There are at least two pathways by which CO is produced endogenously. A
minor pathway is through NADPH-dependent enzymatic peroxidation of
microsomal lipids, with the predominant source of endogenous CO being
the NADPH-dependent oxidative heme destruction catalyzed by heme
oxygenase (HO) isozymes (11). Cleavage of heme by HO results in the
production of CO and biliverdin and release of iron. Two isoforms of HO
have been identified and designated as HO-1 and HO-2 (14, 27). The two
isoforms differ in their regulatory mechanisms. HO-1 is inducible by
many agents and stimuli, including metal ions, heme, organic solvents,
hormones, bacterial toxins, neoplasms, and alkylating agents (11, 14).
The only inducers of HO-2 identified to date are adrenal
glucocorticoids (30).
The potential role for CO as a cellular messenger has recently received
considerable attention. CO is known to increase intracellular guanosine
3',5'-cyclic monophosphate (cGMP) levels in many cell types
(1-3, 28). In the brain, CO has been postulated to play a role in
long-term potentiation (LTP) and in the regulation of cGMP levels (13,
26, 28, 31). Inhibition of HO activity prevents the induction of LTP in
guinea pig hippocampal slices, and perfusion of stimulated nerve fibers
with CO enhances synaptic transmission (31). In mouse and rat
hippocampal slices, inhibition of HO activity inhibits the induction of
LTP and reverses established LTP (26). In olfactory neurons, inhibition
of HO activity reduces endogenous levels of cGMP (28). In addition, in
isolated rabbit corneal epithelial cells, CO increases the open
probability of a
non-Ca2+-dependent
K+ channel and increases
intracellular cGMP levels (22). Other systems in which CO function has
been suggested include regulation of carotid body sensory activity in
the rat and regulation of the release of corticotropin-releasing
hormone and gonadotropin-releasing hormone (9, 18, 19).
There is also a growing body of evidence that suggests a role for CO as
a messenger in the gastrointestinal tract. In the opossum, CO relaxes
the internal anal sphincter and inhibition of HO suppresses
nerve-evoked nonadrenergic, noncholinergic relaxation of the sphincter
(21). In the feline lower esophageal sphincter, HO-2 is present in
neuronal cell bodies and exogenous CO evokes a concentration-dependent
relaxation of the sphincter (17). In human jejunal circular smooth
muscle cells, exogenous CO stimulates an outward
K+ current and a leak current,
hyperpolarizes the membrane potential, and elicits transient membrane
hyperpolarizations (7).
Central to the hypothesis that CO is a physiological messenger in
smooth muscle is the ability of cells in the gastrointestinal tract to
produce CO. CO may be produced from neurons, smooth muscle cells, or
other cells normally found in the smooth muscle layers. CO produced
from neurons may act as an autocrine messenger, regulating neuronal
electrical activity and subsequently smooth muscle contractile activity, or as a paracrine messenger, acting on adjacent cells such as
smooth muscle. Alternatively, CO may be produced by smooth muscle cells
and may act as a local control mechanism for smooth muscle electrical
and mechanical activity. Therefore, the aims of this study were to
examine the distribution of HO in the canine jejunum and to determine
the effects of exogenous CO on jejunal circular smooth muscle cells.
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METHODS |
Immunostaining for HO.
The use of canine jejunum was approved by the Institutional Animal Care
and Use Committee. A piece of jejunum measuring ~1 × 1 cm,
containing the entire thickness of the intestinal wall, was cut from
the jejunum of four dogs just distal to the ligament of Treitz. The
tissues were fixed by overnight immersion in 4% paraformaldehyde in
0.1 M phosphate buffer, pH 7.4, at 4°C. The tissues were then
rinsed several times in phosphate-buffered saline (PBS), immersed in
30% sucrose in PBS overnight at 4°C, and then quick-frozen at
40°C. From each tissue, 20 sections (20 µm thick) were cut
on a cryostat, thawed, and mounted onto chrome alum-coated glass
slides. In the sections, the longitudinal muscle layer was in cross
section, and the circular muscle was cut tangential to its long axis.
Alternate consecutive sections were immunostained for HO-1 or HO-2,
using an indirect immunofluorescence procedure, as follows. All
incubations were done at room temperature and in a moist chamber.
Sections were preincubated with 10% normal goat serum (NGS) in PBS
containing 0.3% Triton X-100 for 45 min, rinsed in PBS, and then
incubated overnight in rabbit antisera to HO-1 or HO-2 diluted 1:500 to
1:1,000 in PBS containing 5% NGS and 0.3% Triton X-100. Sections were
again rinsed in PBS and incubated with rhodamine-conjugated goat
anti-rabbit immunoglobulin G diluted 1:80 in PBS containing 1.25% NGS
and 0.3% Triton X-100 for 90 min. Sections were rinsed with PBS and
then mounted and coverslipped in PBS-glycerol (1:1) containing an
antifade reagent (Slow Fade; Molecular Probes, Eugene, OR) and examined
with a Zeiss Axiophot fluorescence microscope equipped with a rhodamine filter set. As controls, nonimmune rabbit serum was used in place of
the primary antiserum, and HO-2 antibody was preabsorbed with recombinant rat HO-2 protein (100 mg/ml). Immunolabeling was not observed in these controls. Preabsorption of HO-2 antibody with recombinant rat HO-1 protein did not diminish immunolabeling.
In situ hybridization.
In situ hybridization for HO-1 and HO-2 was carried out as detailed by
Ewing (4). Tissues were fixed and embedded in paraffin, and sections 5 µm thick were obtained. Tissue was cleared of paraffin with xylene
and rehydrated by sequential equilibration with graded ethanol
solutions (99-50%) and was then denatured by immersion in 0.2 N
HCl for 20 min at 25°C. Tissue was enzymatically deproteinated by
incubation (15 min, 37°C) with proteinase K and acetylated in 0.1 M
triethanolamine, pH 8, containing 0.25% (vol/vol) acetic anhydride (5 min). Acetylated tissue was dehydrated with graded ethanol
(50-90%) and then prehybridized in buffer containing 50% (vol/vol) deionized formamide, 4× SSC (1× SSC is 0.15 M
NaCl and 0.015 M sodium citrate, pH 7.0), 1% Denhardt's solution,
0.25 mg/ml yeast tRNA, 10% dextran sulfate, and 0.5 mg/ml
heat-denatured salmon sperm DNA. Tissue was hybridized with 2 ng/ml of
the appropriate digoxigenin-labeled (sense and antisense) probe under
parafilm coverslips in a humidified chamber for 16 h at 37°C.
For detection of digoxigenin-labeled HO-2, tissue was rinsed briefly in
100 mM tris(hydroxymethyl)aminomethane (Tris) buffer and blocked with
Tris buffer containing 2% normal sheep serum and 0.3% (vol/vol)
Triton X-100 (30 min, 25°C). Blocker was replaced with a 1:500
dilution of antidigoxigenin antibody conjugated to alkaline
phosphatase, and tissue was incubated with primary antibody at 25°C
for 1 h in a humidified atmosphere. Antibody-antigen complexes were
visualized by incubation of slides in development buffer containing
0.41 mM nitroblue tetrazolium, 0.41 mM 5-bromo-4-chloro-3-indolyl phosphate, and 0.024% (wt/vol) levamisole for up to 16 h in the dark.
Oligonucleotide antisense probes used for in situ hybridization
histochemical studies were complimentary to HO-1 or HO-2 cDNA nucleotides (4, 23). The appropriate sense oligonucleotide sequences
were used as negative controls in these studies. Oligonucleotide probes
for in situ hybridization histochemistry were 3' end-labeled with
digoxigenin-11-dUTP by a tailing reaction using terminal transferase
and were further purified by ethanol precipitation.
Whole cell current measurements.
Single isolated jejunal circular smooth muscle cells were obtained from
adult mongrel dogs of either sex. Each dog was euthanized with an
overdose of barbiturate (45 mg/kg), and a 10-cm piece of jejunum was
removed just distal to the ligament of Treitz. The dissociation
procedures used to obtain single relaxed circular smooth muscle cells
were as previously described (6, 8). In brief, full-thickness strips of
jejunum were pinned to the floor of a dissecting dish, and incisions
were made parallel to the longitudinal muscle axis extending to, but
not into, the circular muscle layer. The serosa and longitudinal muscle
layer were removed, leaving the circular muscle and submucosa.
Incisions were next made parallel to and through the circular muscle
axis. Strips of circular muscle were gently peeled off of the
submucosa, placed in modified Hanks' solution (Sigma H8389), and cut
into 2-mm pieces. They were placed in 8 ml of Hanks' solution
containing 15 mg of papain (Sigma P4762) and 3.1 mg of dithiothreitol
(Sigma D0632) and gently stirred for 25 min at 37°C. After
centrifugation to remove the enzyme solution, the tissue was
transferred to fresh Hanks' solution and mechanically dissociated at
37°C to obtain single relaxed circular smooth muscle cells.
Patch-clamp recordings were made using an Axopatch 200 voltage clamp
amplifier connected to a Digidata 1200, driven by pClamp software (Axon
Instruments, Foster City, CA). Whole cell recordings were obtained
using Kimble KG-12 glass pulled on a P-80 puller (Sutter Instruments,
Novato, CA). Electrodes were coated with Sylgard (184; Dow Corning,
Midland, MI) and fire polished to a final resistance of 3-5 m
.
Unless otherwise stated, all records were obtained using the
amphotericin perforated patch technique (20). Five runs were averaged
for each recording. Records were sampled at 2 kHz and filtered at 1 kHz. Data were analyzed using Clampfit or custom macros in Excel
(Microsoft, Redmont, WA). All patch-clamp recordings were made at room
temperature (24°C), and data are reported as means ± SE. Paired
t-test was used to determine statistical significance. P < 0.05 was considered significant.
cGMP measurements.
cGMP was measured using a radioimmunoassay technique as previously
reported (22). Freshly isolated canine jejunal circular smooth muscle
cells were counted, centrifuged for 7 min at 180 g, and resuspended in 1 ml of normal
Ringer solution containing 0.1 mM 3-isobutyl-1-methylxanthine with or
without 1% CO for 1 min. The cells were frozen in liquid nitrogen and
homogenized, and 5% trichloroacetic acid was added to precipitate
soluble protein. The samples were centrifuged at 1,500 g for 10 min, and the supernatant was
then extracted with water-saturated ether four times to remove the
trichloroacetic acid. Measurements were made in triplicate for each
dilution.
Materials and solutions.
CO was obtained from Scott Specialty Gases (Troy, MI). Quinidine,
amphotericin B, oligo(dT) cellulose, deoxyribonuclease I, proteinase K,
Triton X-100, yeast tRNA, dextran sulfate, 4-chloro-1-naphthol, and
paraformaldehyde were obtained from Sigma (St. Louis, MO), 8-bromo-cGMP
(8-BrcGMP) was from Boehringer Mannheim (Indianapolis, IN), and
KT-5823 was from Calbiochem (San Diego, CA). Copper
protoporphyrin IX (CuPP-IX) was obtained from Porphyrin Products
(Logan, UT). HO-1 and HO-2 antisera and recombinant rat HO-1 and HO-2
protein were obtained from StressGen Biotechnology (Victoria, BC,
Canada). Whole cell recordings were made using the following solution
in the pipette (in mM): 25 KCl, 125 potassium methanesulfonate, 2.54 CaCl2, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid; pH was adjusted to 7.00. Smooth muscle cells were bathed in
normal Krebs solution of the following composition (in mM): 137.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 15.5
, 1.2 H2PO4,
and 11.5 glucose, adjusted to pH 7.35.
For in situ hybridization the following reagents were used: standard
fixative, which consisted of 4% (wt/vol) paraformaldehyde in 0.1 M
phosphate buffer containing 1.5% (wt/vol) sucrose; 2× SSC; and
proteinase K solution, which consisted of 10 mg/ml proteinase K in PBS.
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RESULTS |
Distribution of HO isozymes in the canine jejunum.
Immunoreactivity for HO-2 was observed in a select population of nerve
cell bodies of both myenteric and submucous plexuses in tracts
interconnecting ganglia in a population of nerve fibers in the circular
but not longitudinal muscle layers (Fig.
1). In the ganglia of the myenteric and
submucosal plexuses some nerve cell bodies fluoresced intensely for
HO-2, whereas others fluoresced more faintly or at background levels
only. Because we did not use a marker to label all nerve cell bodies,
we were unable to obtain an accurate percentage of nerve cell bodies
containing HO-2. However, HO-2-containing nerve cell bodies were noted
in all ganglia examined. Immunoreactivity for HO-2 was also observed in
epithelial cells of the jejunal mucosa (data not shown). In contrast,
no HO-1-immunoreactive structures were noted in the tissue
preparations. HO-2 immunoreactivity was not observed in smooth muscle
with the HO antibodies used.

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Fig. 1.
Immunostaining for heme oxygenase-2 (HO-2) in full-thickness sections
(except mucosa) of wall of canine jejunum. Longitudinal muscle (lm)
layer was cut in cross section; circular muscle (cm) layer was cut
tangentially. A: myenteric plexus.
HO-2 immunoreactivity in a subpopulation of nerve cell bodies (arrows)
and fibers interconnecting ganglia (arrowhead).
B: HO-2 immunoreactivity in nerve
fibers (arrowheads) in inner circular muscle layer.
C: submucous plexus. HO-2
immunoreactivity in nerve cell bodies and nerve fiber strands
interconnecting ganglia. Calibration bar (100 µm) applies to all
panels.
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However, HO-2 mRNA was present in both circular and longitudinal smooth
muscle cells (Fig. 2). The message was
detected throughout the circular smooth muscle layer, but in the
longitudinal smooth muscle layer there was staining in a band of smooth
muscle cells at the outer margin of the longitudinal smooth muscle
layer (Fig. 2, A and
B). The significance of the
differential distribution pattern of HO-2 mRNA in longitudinal smooth
muscle is unknown. HO-2 mRNA was also present in a select population of
nerve cell bodies of the enteric plexus. No staining for HO-1 mRNA was
detected in the wall of the jejunum (data not shown).

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Fig. 2.
In situ hybridization localization of HO-2 mRNA in canine jejunum.
A and
B: whole wall thickness
showing positive staining for HO-2 mRNA using antisense probe
(A) in circular smooth
muscle layer and in outer region of longitudinal smooth muscle, and
absence of staining using HO-2 sense probe
(B). Magnification, ×4.
C and
D: higher magnification (×10) of
circular smooth muscle layer shown in
A and
B. E
and F: isolated circular smooth muscle
cells (×100) showing positive staining using HO-2 antisense probe
(E) and negative staining with HO-2
sense probe (F).
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Effects of exogenous CO on isolated jejunal circular smooth muscle
cells.
CO (1%) increased outward current and hyperpolarized the membrane
voltage. An increase in outward current was observed in 19 of 23 cells,
with an increase of 285 ± 86% (n = 23, P < 0.05). The "resting"
membrane potential was
37.2 ± 1.7 mV. This value is similar
to that previously reported in isolated jejunal circular smooth muscle
cells (8). The increase in outward current was accompanied by an
8.5 ± 2.9 mV membrane hyperpolarization. An example of
the effect of 1% CO on an isolated canine jejunal circular smooth
muscle cell is shown in Fig. 3. CO (1%)
increased outward current by 255% in this cell (Fig.
3B). Figure
3C shows the current-voltage relationships of the whole cell currents. The membrane potential of the
cell was
34 mV when bathed in normal Krebs solution. Changing the bath solution to CO (1%) resulted in an increase in outward current and a shift in the membrane potential from
34 to
50 mV. The difference current was obtained by subtracting the
control current from the stimulated current and represents the portion of the current selectively activated by CO. The difference current reversed at
78 mV, close to the equilibrium
potential for K+ (
82 mV), suggesting a
specific effect of CO on K+
current. The leak current, defined as an ohmic current that reversed at
0 mV, did not change in this cell. However, the leak current increased
by 20 ± 18% in the 23 cells studied
(P < 0.05).

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Fig. 3.
Effect of carbon monoxide (CO) on an isolated canine jejunal circular
smooth muscle cell. Whole cell current recorded in normal Krebs
solution (A) increased by 255% when
CO (1%) was present (B).
C: current-voltage relationships show
effects of CO on outward current and membrane potential. CO increased
outward current and shifted membrane potential from 34 to
50 mV. Difference current was obtained by subtracting control
current from CO-stimulated current and represents portion of current
selectively activated by CO. Difference current reversed at 78
mV, near equilibrium potential for K+ ( 82 mV),
suggesting an effect of CO on K+
current. No change in leak current, defined as an ohmic current that
reversed at 0 mV, was noted in this cell.
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To further examine whether the increase in outward current was due to
an increase in K+ conductance, the
effects of the K+ channel blocker
quinidine on the CO-stimulated current were examined. Quinidine (50 µM, n = 4) blocked the effects of CO
on outward current. An example of the effect of quinidine on the
CO-stimulated outward current is shown in Fig.
4. CO (1%) increased peak outward current
(Fig. 4B) by 247%. Addition of 50 µM quinidine blocked the outward current (Fig.
4C). Figure
4D shows the current-voltage relationships obtained from the whole cell currents. CO shifted the
reversal potential from
35 to
47 mV. Quinidine (50 µM)
blocked the outward current and shifted the reversal potential from
47 to
2 mV. To determine whether the increase in
K+ current was due to activation
of charybdotoxin-sensitive
Ca2+-activated
K+ channels known to be present in
canine jejunal circular smooth muscle, exogenous CO (1%) was applied
to cells in the presence of charybdotoxin (100 nM). In the presence of
charybdotoxin CO increased outward current by 198 ± 50%
(P < 0.05, n = 4), suggesting that
charybdotoxin-sensitive
Ca2+-activated
K+ channels were not involved in
the increase in K+ current evoked
by CO (Fig. 5) and that CO selectively
activated the quinidine-sensitive
K+ current that determines the
membrane potential (6, 8).

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Fig. 4.
Effect of the K+ channel blocker
quinidine (Quin) on CO-stimulated outward current.
A: whole cell current obtained in
Krebs solution. B: effect of CO (1%)
on outward current. C: effect of
K+ channel blocker quinidine (50 µM) on CO-stimulated outward current. Note block of outward current
by quinidine. D: current-voltage
relationships obtained from whole cell currents. CO increased outward
current and shifted reversal potential from 35 to 47 mV.
Quinidine (50 µM) blocked outward current and shifted reversal
potential from 47 mV to 2 mV. Results suggest that CO
activated the quinidine-sensitive
K+ current that determined the
membrane potential.
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Fig. 5.
Effect of CO on an isolated canine jejunal circular smooth muscle cell
in presence of charybdotoxin. Whole cell current recorded in normal
Krebs solution (A) increased by
136% when CO (1%) was applied in presence of charybdotoxin (100 nM)
to block Ca2+-activated
K+ channels
(B), suggesting that
Ca2+-activated
K+ channels were not involved in
the mechanism of action of CO. C:
current-voltage relationships show effects of CO on outward current and
membrane potential. CO increased outward current and shifted membrane
potential from 30 to 51 mV. Difference current, obtained
by subtracting control current from CO-stimulated current, reversed at
67 mV and represents portion of current selectively activated by
CO.
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Effects of long-term application of CO on outward current.
In isolated human jejunal circular smooth muscle cells, long-term
application (minutes) of CO evokes cyclic changes in outward current
and in membrane potential (7). To determine whether the same effects
were also present in isolated canine jejunal circular smooth muscle
cells, whole cell outward currents were measured over a period of at
least 15 min. In six of six cells tested, CO evoked cyclic changes in
whole cell outward current. An example is shown in Fig.
6. The figure shows peak current measured at +40 mV and recorded at 1-s intervals. Marked oscillations in outward
current were seen in the presence of CO, in contrast to the control
tracing obtained in Krebs solution in the absence of CO. Because the
oscillations were suggestive of spontaneous transient outward
oscillations in current evoked by transient release of intracellular
Ca2+, changes in intracellular
Ca2+ were monitored in a separate
series of experiments. Isolated canine jejunal circular smooth muscle
cells were loaded with fura 2-acetoxymethyl ester, and the fluorescence
emitted at excitation wavelengths of 340 and 380 nm was recorded. No
effect of CO (1%) on intracellular
Ca2+ levels was noted. The 340:380
nm ratio was 1.4 ± 0.4 before and 1.36 ± 0.5 after application
of CO (P > 0.05, n = 6). Also, in suspensions of
isolated canine jejunal circular smooth muscle cells loaded with indo
1, no changes in intracellular
Ca2+ were seen using a
fluorescence-activated cell sorter. These experiments, however, do not
rule out localized increases in intracellular Ca2+ below the resolution of these
techniques. To determine whether the increase in outward current
observed with CO was dependent on intracellular
Ca2+, traditional whole cell
experiments were performed with 2 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid in the recording pipette. After access to the cell was obtained, the cell interior was allowed to equilibrate with the pipette solution
for 10 min. Application of CO (1%) increased outward current by 58 ± 40% (n = 6, P < 0.05) and hyperpolarized the
membrane potential by 5.7 ± 3 mV
(n = 6). As the increase in current in the standard whole cell configuration was less than that seen in
amphotericin perforated patches, the data suggest that intracellular Ca2+ may be necessary for the full
effects of CO, although washout of other intracellular messengers
necessary for the effects of CO was also a possibility.

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Fig. 6.
Cyclic changes in whole cell outward current evoked by application of
CO (1%). Shown is peak current measured at +40 mV and recorded at 1-s
intervals. Oscillation in outward current seen in presence of CO is in
contrast to the absence of any oscillation when cell was treated with
normal Krebs solution.
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Effect of CO on intracellular cGMP.
CO is among the endogenous factors known to activate soluble guanylyl
cyclase (24) and therefore to stimulate the production of cGMP in many
cell types (1-3, 28). The effect of CO (1%) on intracellular cGMP
levels is shown in Fig. 7. CO (1%)
increased cGMP levels from 86 ± 32 to 178 ± 70 pmol/106 cells
(n = 5, P < 0.05). To determine whether
exogenous cGMP could mimic the effects of CO on outward current,
8-BrcGMP (membrane-permeable form of cGMP) was applied to the bathing
solution. 8-BrcGMP (2 mM) increased whole cell current (94.2 ± 37%, n = 10, P < 0.05) and hyperpolarized (5.4 ± 2.7 mV, n = 10) the membrane
potential (Fig. 8). If the effects of CO
were mediated solely through cGMP, the further addition of CO would not
be expected to further affect the outward current or membrane
potential. Addition of 1% CO to the cGMP-stimulated current further
increased the whole cell current, suggesting that in amphotericin
perforated patch recordings the effects of CO may not be solely
mediated through cGMP (Fig. 9). Cyclic
increases in the whole cell current accompanied by membrane hyperpolarization were also noted with cGMP (data not shown).

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Fig. 7.
Effect of CO on intracellular cGMP concentration measured by
radioimmunoassay. Freshly dispersed cells in Krebs solution with 0.1 mM
3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor used to
prevent cGMP breakdown, were divided into 2 samples
(n = 5). 1% CO significantly
increased cGMP levels from 86 ± 32 to 178 ± 70 pmol/106 cells
(P < 0.05).
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Fig. 8.
Effect of cGMP on whole cell outward current membrane potential.
A: whole cell current recorded in
Krebs solution. B: whole cell current
recorded 8 min after application of 8-bromo-cGMP (8-BrcGMP; 2 mM).
Current increased by 85%. C:
current-voltage relationships for whole cell currents with a 17 mV
hyperpolarization in membrane potential in presence of cGMP.
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Fig. 9.
Effect of CO on cGMP-stimulated outward current.
A: whole cell current recorded in
Krebs solution. B: whole
cell current recorded 7 min after application of 2 mM 8-BrcGMP. Current
increased by 30%. C: effect of CO
(1%) on cGMP-stimulated current. Outward current further increased by
65%. D: current-voltage relationships
for whole cell currents. cGMP increased outward current and
hyperpolarized membrane potential from 30 mV to 35 mV. CO
further increased outward current and hyperpolarized membrane potential
to 43 mV.
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Effect of KT-5823.
The effects of KT-5823, an inhibitor of cGMP-dependent protein kinase,
on outward current and membrane potential were studied to further
determine whether the hyperpolarization and increase in outward current
seen on application of CO to the bath was due to cGMP or cGMP-dependent
protein kinase. Cells were patch clamped in the standard whole cell
configuration with KT-5823 (1 µM) in the recording pipette solution.
After establishing access, control currents were observed for 10 min to
allow KT-5823 to diffuse into the cell. CO (1%) was then added to the
bath solution. The whole cell outward current increased by 7 ± 5%, and the membrane potential hyperpolarized by 0.3 ± 1.6 mV (n = 4, P > 0.05, data not shown). The data
suggest that under standard whole cell recording conditions the
increase in outward current and hyperpolarization evoked by CO was
mediated primarily through a cGMP-dependent protein kinase.
Inhibition of HO.
A major hurdle to the study of the role of CO as a physiological
messenger is the absence of a specific blocker of HO. Zinc protoporphyrin IX has been used in several studies to inhibit HO;
however, zinc protoporphyrin IX is also known to directly inhibit
guanylyl cyclase (16) and to inhibit the actions of vasoactive
intestinal polypeptide and atrial natriuretic peptide, both smooth
muscle relaxants, through a non-HO pathway (16). Recently, CuPP-IX has
been proposed as an inhibitor of HO with no effect on guanylyl cyclase
(19, 29). Therefore, the effects of CuPP-IX on the whole cell current
of isolated canine jejunal circular smooth muscle cells were
determined. On addition to the bath, CuPP-IX (10 µM) evoked an
immediate increase in outward current of 259 ± 94%
(P < 0.05, n = 4). Prolonged exposure to CuPP-IX
(30 min) resulted in a 25.3 ± 9%
(P < 0.05) decrease in current over
the maximal current recorded in the presence of CuPP-IX. However, the
whole cell outward current did not return to baseline in all four
cells. Washout of CuPP-IX (30 min) resulted in a 17 ± 6% increase
in current (Fig. 10). The results may
suggest a potential role for endogenous CO as an intracellular
messenger in smooth muscle; however, due to the initial stimulatory
effect of CuPP-IX, the role of CO as a messenger remains uncertain
until more specific HO or CO inhibitors become available.

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Fig. 10.
Effect of copper protoporphyrin IX (CuPP-IX; 10 µM) on whole cell
current recorded from an isolated canine jejunal circular smooth muscle
cell. Peak current (at +40 mV) is plotted against time. Prolonged
exposure to CuPP-IX decreased outward current by 18% over the maximal
outward current recorded in presence of CuPP-IX. Washout of CuPP-IX
resulted in a 36% increase in outward current.
|
|
 |
DISCUSSION |
We have shown in canine jejunum that HO-2, an enzyme whose activity
leads to the endogenous production of CO, was present in neuronal cell
bodies in a population of neurons in the myenteric and submucous
plexuses, in nerve fibers innervating the circular smooth muscle layer,
and in smooth muscle cells as well as in epithelial cells in the
mucosa. We have also shown that exogenous CO stimulated intracellular
cGMP production and activated a K+
conductance in circular smooth muscle cells, resulting in membrane hyperpolarization.
Endogenous production of CO as a byproduct of heme metabolism has been
well known for many years (10). However, the potential role of CO
generated by HO activity as a physiological messenger was only recently
postulated (12, 15, 25). The data presented in this study indicate that
the machinery for CO production (HO) was present in both neurons and
smooth muscle, suggesting that CO may function as both a paracrine and
an autocrine messenger in the gastrointestinal tract. Exogenous CO
resulted in an increase in cGMP production in isolated jejunal circular
smooth muscle cells and activated a
K+ current. Both effects are
expected to result in smooth muscle relaxation, suggesting that CO may
act as an inhibitory messenger in the gastrointestinal tract.
The marked variation in the distribution of HO-2 among neuronal cell
bodies in the canine jejunum, with some nerve cell bodies staining
deeply for HO-2 while adjacent nerve cell bodies in the same ganglion
show little or no staining, would argue in favor of a specific role for
HO-2, perhaps in CO production, as opposed to reflecting a ubiquitous
distribution of the enzyme. The two known constitutive isoforms of HO
(HO-1 and HO-2) differ in their molecular weight, structure, and
response to inducers. No evidence for specific immunostaining with HO-1
was seen in canine jejunal myenteric nerve cell bodies or in smooth
muscle. HO-1, under normal conditions, is expressed at low levels in
all tissues but the spleen. However, HO-1 activity in the rat can be
increased 10- to 100-fold by a variety of agents, including heme and
metal ions (10, 11). In the control state it appears that HO-2 is the predominate isoform in the canine jejunum; however, the presence of
inducible HO-1 in canine jejunum at levels below the detection limits
of our technique is also possible. This is in fact likely, as molecular
biological techniques have demonstrated the presence of induced HO-1 in
all tissues tested. For example, in the brain, levels of HO-1 are below
the detection limit of Western immunoblotting, but HO-1 can be induced
by hyperthermia and has been shown to be present by
immunohistochemistry (5).
The effects of CO on peak whole cell current and membrane potential
were similar to results previously reported in the human jejunum. In
isolated human jejunal circular smooth muscle cells, 1% CO increases
outward current by 175 ± 40% and hyperpolarizes the membrane
potential by 15.6 ± 3.6 mV (7), similar to the 285 ± 86%
increase in whole cell current and 8.5 ± 2.9 mV hyperpolarization of the membrane potential reported in this study on isolated canine jejunal circular smooth muscle cells. In both canine and human cells,
long-term application of CO evoked cyclic changes in outward current
and membrane potential, suggesting a similar mechanism of action for CO
in both species.
In summary, in the canine jejunum, HO-2, a source for endogenous CO,
was localized in a defined population of neuronal cell bodies, in nerve
fibers, and in smooth muscle cells. Exogenous CO evoked an increase in
outward K+ current accompanied by
membrane hyperpolarization in isolated canine jejunal circular smooth
muscle and increased cGMP levels. The data suggest that CO is
endogenously produced in the canine jejunum in both nerves and smooth
muscle, raising the possibility that CO may be an autocrine and
paracrine messenger in the gastrointestinal tract.
 |
ACKNOWLEDGEMENTS |
We thank Joan Rae, Gary Stoltz, and Xiaodan Zhao for technical
assistance and Jan Applequist for secretarial assistance.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
DK-17238, EY-03282, EY-06005, and ES-03968 and by an American Gastroenterological Association industry research scholar award.
Present address of X. Liu: Dept. of Physiology, Univ. of Nevada School
of Medicine, Anderson Medical Sciences Bldg., Reno, NV 89557-0046.
Address for reprint requests: G. Farrugia, 8 Guggenheim, Dept. of
Physiology and Biophysics, Mayo Clinic and Mayo Foundation, 200 First
St. SW, Rochester, MN 55905.
Received 14 April 1997; accepted in final form 3 November 1997.
 |
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