Vol. 276, Issue 1, G138-G145, January 1999
Mechanism of inhibition of VIP-induced LES relaxation by heme
oxygenase inhibitor zinc protoporphyrin IX
Satish
Rattan,
Ya-Ping
Fan, and
Sushanta
Chakder
Department of Medicine, Division of Gastroenterology and Hepatology,
Jefferson Medical College, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107
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ABSTRACT |
The putative heme
oxygenase inhibitor zinc protoporphyrin IX (ZnPP IX) is known to exert
diverse actions, including inhibitory action on smooth muscle
relaxation by vasoactive intestinal polypeptide (VIP). The studies were
performed in the opossum lower esophageal sphincter (LES) smooth muscle
to determine the site of the inhibitory action of ZnPP IX in the smooth
muscle relaxation by VIP. We also examined the effect of a direct
Gs protein activator, cholera toxin (CTX), known to stimulate adenylate cyclase (AC). CTX caused relaxation of the LES smooth muscle by its action directly at the
smooth muscle cells. The convergence of the common mechanisms of
actions of VIP and CTX on AC was determined by the suppression of their
effects by the AC inhibitor and CTX desensitization. ZnPP IX caused
attenuation of the LES smooth muscle relaxation by VIP but not by CTX.
ZnPP IX but not zinc deuteroporphyrin IX caused significant inhibition
of VIP binding to the membrane receptor. We conclude that ZnPP IX
attenuates VIP-induced LES smooth muscle relaxation by inhibition of
VIP binding to G protein-coupled receptors linked to AC at a point
proximal to G protein activation.
vasoactive intestinal polypeptide; lower esophageal sphincter; inhibitory neurotransmission; nitric oxide synthase; G protein
coupled
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INTRODUCTION |
MOST OF THE NEUROHUMORAL receptor interactions leading
to a physiological action occur in the following steps. The binding of
an agonist to the specific receptor causes change in the receptor conformation, followed by activation of the specific G protein, interaction with the specific enzyme associated with second messenger, interaction with ion channels or other target protein, and finally the
specific action. The specific action in the case of the
gastrointestinal smooth muscle may either be a relaxation or
contraction. The neurohumoral substance examined in the present study
is vasoactive intestinal polypeptide (VIP), which causes relaxation of
the lower esophageal sphincter (LES) by the activation of receptors
located on the smooth muscle cell membranes.
Carbon monoxide (CO) may be produced endogenously from heme by its
interaction with heme oxygenase (HO) (26). Two types of HO have been
recognized, HO-2 primarily in neural tissues and HO-1 in nonneural
tissues. Zinc protoporphyrin IX (ZnPP IX) has been suggested to be a
selective inhibitor of HO in a number of systems (26). Although the
exact role of CO in the gastrointestinal smooth muscle is not known, it
has been shown to cause a direct relaxation in a number of smooth
muscle preparations (16, 31, 43), including the LES (28) and the
internal anal sphincter (IAS) (31). Progress on the role of the HO
pathway in inhibitory neurotransmission has been limited by the lack of
selective HO inhibitors.
The putative HO inhibitor ZnPP IX has been recognized to have multiple
actions especially in different smooth muscles, in addition to HO
inhibition (14, 21). Because of this, caution should be exercised while
using this agent primarily to determine the role of the HO pathway.
Among other actions, ZnPP IX has been suggested to cause the blockade
of VIP-induced relaxation of the IAS (31) as well as LES (28) and other
smooth muscles. The exact site of action of ZnPP IX in blocking VIP
response has not been investigated. To analyze this issue, specific
tools to block or activate different steps along VIP receptor
interaction, direct activator of
Gs protein (cholera toxin or CTX),
inhibitors of adenylate cyclase (AC), and CTX desensitization were
employed. There is substantial evidence to suggest that VIP-induced
smooth muscle relaxation is primarily mediated by the G protein-coupled receptor stimulation of AC (4, 35).
The main purpose of the present investigation therefore was to
determine the mechanism of the inhibitory action of ZnPP on the LES
smooth muscle relaxation caused by VIP. In the process, direct receptor
binding studies with VIP before and after ZnPP IX were also performed.
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MATERIALS AND METHODS |
Preparation of smooth muscle strips.
The LES smooth muscle strips from opossums (Didelphis
virginiana) of either sex were prepared for the
recording of isometric tension as described previously (32). Briefly,
after receiving anesthesia with pentobarbital (40-50 mg/kg ip) the
animals were killed by exsanguination, and the LES along with a section
of the esophagus and stomach was isolated and transferred to oxygenated (95% oxygen plus 5% carbon dioxide) Krebs physiological solution of
the following composition (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4,
25 NaHCO3, and 11.10 glucose. The
LES was carefully freed of all extraneous tissues, including the large
blood vessels, opened, and pinned flat with the mucosal side up on a
dissecting tray containing oxygenated Krebs solution. The mucosal and
submucosal layers were removed by sharp dissection, and LES circular
smooth muscle strips (1 × 10 mm) were prepared as described
previously (32).
Measurement of isometric tension.
The smooth muscle strips were tied at both ends with silk sutures (6-0;
Ethicon, Sommerville, NJ) and transferred to 2-ml muscle baths
containing oxygenated Krebs solution (37°C). One end of the muscle
strip was anchored at the bottom of the muscle bath and the other end
was attached to a force transducer (model FTO3; Grass Instruments,
Quincy, MA) for the measurement of isometric tension on a Dynograph
recorder (model R411; Beckman Instruments, Schiller Park, IL). The
muscle strips were stretched initially at 1 g of tension and then
allowed to equilibrate for at least 1 h with regular washings at 20-min
intervals. Only the strips that developed spontaneous steady tension
and relaxed in response to electrical field stimulation (EFS) were
used. The optimal length and the baseline of the smooth muscle strips
were determined as described previously (27).
NANC nerve stimulation with EFS.
EFS was delivered from a Grass stimulator (model S88; Quincy, MA)
connected in series to a Stimu-Splitter II (Med-Lab Instruments, Loveland, CO). The Stimu-Splitter served an important purpose to
amplify and measure the stimulus intensity using the optimal stimulus
parameters for the neural stimulation (12 V, 0.5-ms pulse duration,
200-400 mA, 4-s train) at varying frequencies of 0.5 to 20 Hz. The
electrodes used for the EFS consisted of a pair of platinum wires fixed
at both sides of the smooth muscle strip. The parameters of EFS stated
above are known to selectively cause relaxation of the LES smooth
muscle via the activation of nonadrenergic noncholinergic (NANC)
myenteric neurons.
Drugs and chemicals.
The following chemicals were used in the study: ZnPP IX, isoproterenol
hydrochloride, and N-ethylmaleimide
(NEM) (Aldrich Chemical, Milwaukee, WI); zinc deuteroporphyrin IX 2,4 bis-ethylene glycol (ZnDP IX) (Porphyrin Products, Logan, UT);
NG-nitro-L-arginine
(L-NNA) and sodium nitroprusside
(SNP) (Sigma Chemical, St. Louis, MO), and VIP (Bachem Bioscience,
Torrance, CA); and CTX and 9-(tetrahydro-2-furanyl)-9H-purin-6-amine
(SQ-22536) (Research Biochemicals International, Natick, MA) and EDTA
tetrasodium (Fisher Scientific, Pittsburgh, PA).
125I-VIP (2,000 Ci/mmol) was
obtained from Amersham (Arlington Heights, IL).
VIP and isoproterenol are known to activate AC via
Gs protein coupling (21, 33). CTX
is well known to cause the smooth muscle relaxation by its direct and
selective activation of Gs protein
and AC (22-24, 41). To determine smooth muscle relaxation via the
AC pathway, AC inhibitors SQ-22536 (18) and NEM (11, 27, 32, 38) were used.
All chemicals except ZnPP IX and ZnDP IX were dissolved and diluted in
Krebs solution and prepared fresh on the day of the experiment. Stock
solutions of ZnPP IX and ZnDP IX were prepared by dissolving in 0.2 N
sodium hydroxide and diluting with Krebs solution and kept in the dark.
The pH of ZnPP IX and ZnDP IX solutions was adjusted to 7.4 using 0.2 N
HCl. The final dilution of sodium hydroxide used as solvent for the
porphyrins produced no significant effect on the basal LES tone.
The vials and pipette tips were siliconized while the muscle baths were
treated with 2.5% BSA to prevent the binding of VIP to the surface.
Receptor binding studies.
To determine the influence of ZnPP IX on specific binding of VIP to the
LES smooth muscle receptor, we examined the effects of ZnPP IX on VIP
binding and compared them with those of ZnDP IX. VIP binding
experiments on LES smooth muscle membranes were carried out as
described previously (8). Briefly, after isolation of the LES from the
animals, the smooth muscle was cleaned off the mucosa and other
adhering tissues including the serosa and the small blood vessels. The
tissue was cut into small pieces and homogenized on an ice bath in Tris
buffer (25 mM, pH 7.4) containing 0.32 M sucrose using an Ultraturrax
tissue homogenizer (Tekmar, Cincinnati, OH). The homogenate was
centrifuged at 1,000 g for 20 min
(4°C). The supernatant was transferred to a separate tube and
centrifuged at 50,000 g for 30 min
(4°C). The pellet was resuspended in Tris buffer (25 mM, pH 7.4)
containing 2 mM EDTA and centrifuged at 50,000 g for 30 min. The pellet was washed twice with the same buffer after resuspending and centrifugation in the
same way. The final pellet was suspended in Tris buffer, and aliquots
were stored at 
80°C until used for VIP binding experiments. The protein contents of the membranes were determined by using the
method of Lowry et al. (25) with BSA as the standard.
Binding experiments were carried out in duplicate in Tris buffer (25 mM, pH 7.4) containing BSA (protease free, 1.5%), bacitracin (1 mg/ml), and EDTA (2 mM) in a final volume of 0.2 ml. The membranes were
diluted in the buffer (50 µg/ml final concentration) and incubated
with radiolabeled VIP (45 pM) for 10 min at 30°C with or without
unlabeled VIP. For examining the effects of ZnPP IX or ZnDP IX on VIP
binding to the LES smooth muscle membranes, the membranes were first
incubated with 1 × 10
4 M ZnPP IX or ZnDP IX
for 10 min before performing the binding studies with
125I-VIP in the presence or
absence of different concentrations of unlabeled VIP. The incubation
was stopped by adding 1 ml of ice-cold Tris buffer to the incubates,
and the membrane-bound radioactivity was separated from the unbound
radioactivity by centrifugation at 16,000 g for 5 min. The pellets were washed
again with the addition of 1 ml Tris buffer and recentrifugation. The
radioactivity in the pellets were counted using a gamma counter
(Genesis, Laboratory Technology, Schaumburg, IL). The specific binding
was determined by subtracting the radioactivity remaining in the
presence of 1 × 105 M
unlabeled VIP from the total radioactivity. The nonspecific binding was
~25% of the total binding.
Drug responses.
Pretreatment with ZnPP IX and ZnDP IX (1 × 104 M) for 10 min was used
to examine their effects on the basal LES tone and changes in response
to different agonists. The dose was chosen based on previous data that
showed that ZnPP IX in this dose was maximally effective in blocking HO
activity in the gastrointestinal smooth muscle preparations (28). To
examine the influence of AC inhibitors, the smooth muscle strips were
pretreated with SQ-22536 (1 × 105 to 3 × 104 M) or NEM (1 × 104 M) for 10 min before
testing the effects of EFS, VIP, and CTX. The concentrations of
different antagonists were selected in view of the earlier literature
and our own experiments. The doses examined were relatively selective
against the intended effects.
In some experiments the effect of VIP was examined in the presence of
CTX desensitization. CTX desensitization was achieved by the repeated
administration of the toxin (1 µg/ml) until its effect was
substantially reduced.
All experiments involving ZnPP IX and ZnDP IX were carried out in the
dark. All the agonists were given in a cumulative fashion. Once the
concentration-response curve to an agent was determined, the smooth
muscle strips were washed at least six times and the resting tension
was allowed to recover to the preinjection levels.
Data analysis.
The results are expressed as means ± SE of different experiments.
The fall of the resting LES tension is expressed as the percentage of
maximal potential response (100%) to a supramaximal concentration (5 mM) of EDTA. Statistical significance between different
groups was determined by using paired or unpaired
t-test where applicable, and
P < 0.05 was considered
statistically significant.
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RESULTS |
Influence of HO inhibitors ZnPP IX and ZnDP IX on LES smooth muscle
relaxation by VIP.
The data in Fig. 1 show that ZnPP IX caused
a marked and significant (P < 0.05, n = 5) shift in the dose-response
curve of VIP in causing the LES relaxation. ZnDP IX on the other hand
had a relatively limited effect. The values of the fall in the basal tension of LES with 3 × 107 M VIP before and after
ZnPP IX (1 × 104 M)
were 75.5 ± 7.8 and 25.1 ± 3.9%, respectively
(P < 0.05, n = 5), and the corresponding values
before and after ZnDP IX (1 × 104 M) were 72.2 ± 5.5 and 70.0 ± 3.8%, respectively (P > 0.05, n = 5). However, ZnDP IX did
cause a significant suppression of the LES smooth muscle relaxation in
response to lower concentrations of VIP (1 × 108 to 1 × 107 M). This suppression by
ZnDP IX was significantly less compared with that by ZnPP IX. ZnPP IX
as well as ZnDP IX had no significant effect on the basal tone of the
LES. The basal LES tone before and after ZnPP IX was 1.8 ± 0.1 and
1.8 ± 0.1 g, and before and after ZnDP IX it was 2.2 ± 0.1 and
2.2 ± 0.1 g, respectively (P > 0.05, n = 5).
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Fig. 1.
Influences of heme oxygenase (HO) inhibitors zinc protoporphyrin IX
(ZnPP IX) and zinc deuteroporphyrin IX (ZnDP IX) (1 × 104 M) on percent fall in
lower esophageal sphincter (LES) tension by vasoactive intestinal
polypeptide (VIP). Data show that both ZnPP IX and ZnDP IX caused
significant attenuation of VIP responses
(* P < 0.05, n = 5). However, compared with ZnPP
IX, ZnDP IX had markedly less effect in blocking action of VIP. Effect
of VIP in concentrations higher than 1 × 107 M was not significantly
affected by ZnDP IX. Comparison of entire VIP dose-response curves
revealed that differences were significant from control only in case of
ZnPP IX. Asterisk to right of curve in this and other figures indicates
entire curve is significantly different from control. Asterisks above
individual values indicate significant difference from
counterpart controls.
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We have substantial data to show that in contrast to its actions on
VIP, ZnPP IX had no significant effect on the LES smooth muscle
relaxation caused by forskolin, SNP, nitric oxide (NO), and CO (14).
Furthermore, the LES smooth muscle contraction caused by bethanechol
was also not modified by ZnPP IX.
Influence of ZnPP IX and ZnDP IX on LES smooth muscle relaxation by
NANC nerve stimulation with EFS.
Neither ZnPP IX nor ZnDP IX had any significant effect on the LES
smooth muscle relaxation by the NANC nerve stimulation with EFS (Fig.
2; P > 0.05, n = 9). The fall in
the basal LES tension with 5 and 10 Hz of EFS in these experiments was
71.5 ± 2.7 and 76.5 ± 2.7%, and in the presence of
ZnPP IX and ZnDP IX these values were 68.8 ± 4.6, 75.7 ± 3.3, 70.7 ± 3.9, and 74.3 ± 3.3%, respectively.
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Fig. 2.
Effects of ZnPP IX and ZnDP IX on fall in LES tension induced by
electrical field stimulation (EFS). None of these HO inhibitors had
any significant effect on EFS-induced LES relaxation
(P > 0.05, n = 9).
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To determine the relationship between the actions of VIP and CTX, the
effects and the site of action of CTX in causing the LES smooth muscle
relaxation were investigated first.
Effects and site of action of CTX on basal LES tone: influences of
L-NNA, TTX, and
-conotoxin.
The role of Gs protein in the
VIP-induced relaxation of the LES smooth muscle and the influence of
ZnPP IX were examined by the use of CTX, which causes direct activation
of Gs protein. CTX caused a
concentration-dependent fall in the basal tension of the LES (Fig.
3). A maximal fall in the basal LES tension
was observed with 2 µg/ml CTX in the muscle bath. The percent fall in
the basal tension of the LES with 2 µg/ml of CTX was 79.6 ± 2.0. The responses of CTX on the LES were not modified by the NO synthase
(NOS) inhibitor L-NNA, the
neurotoxin TTX (1 × 106 M), and the combination
of TTX and the neuronal calcium channel blocker -conotoxin GVIA (1 × 106 M)
(P > 0.05, n = 4; Fig.
4).
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Fig. 4.
Influences of nitric oxide synthase inhibitor
NG-nitro-L-arginine
(L-NNA), neurotoxin TTX, and
neuronal Ca2+ channel blocker
-conotoxin on LES relaxation caused by CTX (2 µg/ml). These
inhibitors caused no significant inhibition of the fall in LES tension
induced by CTX (P > 0.05, n = 4).
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Influence of ZnPP IX on LES smooth muscle relaxation by CTX.
The dose-response curves for CTX showing the percent fall in the basal
tension of the LES, obtained before and after ZnPP IX were found
not to be significantly different (P > 0.05, n = 5). The percent fall in
the basal LES tension with 0.5, 1.0, and 2.0 µg/ml in control
experiments was 65.2 ± 3.7, 72.9 ± 3.4, and 79.6 ± 2.0, respectively. These values in the presence of ZnPP IX were 67.5 ± 2.5, 75.4 ± 3.0, and 81.7 ± 3.0%, respectively.
Influence of AC inhibitors on LES smooth muscle relaxation by VIP
and CTX.
The commonly used AC inhibitor SQ-22536 was found to be relatively
nonselective and ineffective in blocking AC in the LES smooth muscle.
In a different range of concentrations (1 × 105 to 3 × 104 M), previously reported
to be specific for the purpose, SQ-22536 caused a significant fall in
the basal tone of the LES. Additionally, SQ-22536 had no significant
effect on the LES relaxation caused by CTX, known to activate AC.
SQ-22536 (1 × 104 M
and 3 × 104 M) caused
a fall in the basal tension of the LES from 2.2 ± 0.3 to 1.5 ± 0.2, and 1.1 ± 0.3 g, respectively
(P < 0.05, n = 5).
For this reason, studies to examine the role of AC in the LES smooth
muscle relaxation were performed using NEM, previously reported to
cause a significant blockade of the AC pathway (11, 27, 32, 38). NEM (1 × 104 M) caused a
significant suppression of the fall in LES tension by EFS (Fig.
5B) and
isoproterenol (Fig. 5C)
(P < 0.05, n = 5) but had no significant effect
on the relaxant actions of SNP (P > 0.05, n = 5; Fig.
5A). NEM (1 × 104 M) also caused a
significant blockade of the effect of different concentrations of CTX
in causing the LES smooth muscle relaxation (P < 0.05, n = 5; Fig.
6). Likewise, the fall in the basal LES tension by different concentrations of VIP was also significantly blocked by NEM (P < 0.05, n = 5; Fig.
7).
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Fig. 5.
Influence of adenylate cyclase inhibitor
N-ethylmaleimide (NEM) on percent fall
in LES tension caused by sodium nitroprusside (SNP)
(A), EFS
(B), and isoproterenol
(C). Note that NEM caused
significant attenuation of fall in LES tension induced by EFS and
isoproterenol (* P < 0.05, n = 5) but not by SNP
(P > 0.05, n = 5).
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Fig. 6.
Data show that fall in LES tension caused by CTX was significantly
blocked by adenylate cyclase inhibitor NEM (1 × 104 M)
(* P < 0.05, n = 5).
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Fig. 7.
Data show significant blockade of effect of VIP on LES by adenylate
cyclase inhibitor NEM (1 × 104 M)
(* P < 0.05, n = 5).
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The data reported suggest that the actions of both VIP and CTX in
causing the LES smooth muscle relaxation are mediated via the
activation of Gs protein that is
linked to the AC. To further establish the common pathway for their
relaxation, the influence of CTX desensitization on VIP-induced
relaxation of the LES was examined next.
Influence of CTX desensitization on VIP-induced relaxation of LES.
The frequent administration of CTX caused a significant reduction in
its responses. Furthermore, CTX desensitization also caused a
significant reduction in VIP responses in the LES
(P < 0.05, n = 4; Fig.
8), suggesting that the LES relaxation by CTX and VIP follows the same biochemical pathway.
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Fig. 8.
Data demonstrating phenomenon of CTX desensitization (Des.) and its
influence on VIP-induced LES relaxation. CTX desensitization caused
significant reduction in LES relaxation induced by CTX (1 µg/ml) and
VIP (3 × 107 M)
(* P < 0.05, n = 4).
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The site of action of ZnPP IX in suppressing the LES relaxation by VIP
appears to be at a point above the activation of the G protein since
the fall in the LES tension by CTX was not affected by ZnPP IX. The
next focus therefore was to examine the possibility of inhibition of
VIP binding by ZnPP IX at the membrane receptor.
Influence of ZnPP IX on binding of VIP to its receptors in LES.
We compared the effects of different concentrations of unlabeled VIP on
125I-VIP binding to the LES smooth
muscle membranes before and after ZnPP IX and ZnDP IX (1 × 104 M). The data given in
Fig. 9 show that in control experiments, VIP caused a significant and concentration-dependent displacement of
the bound 125I-VIP. Although ZnDP
IX had no significant effect on this control VIP receptor binding
curve, ZnPP IX caused a significant inhibition of the displacement
curve. These data suggest that ZnPP IX interferes with the binding of
VIP to the receptor. Furthermore, the data correspond to the functional
data in Fig. 1 that show the suppression of the VIP-induced fall in the
basal LES tension in the presence of ZnPP IX.
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Fig. 9.
Inhibition of 125I-VIP binding to
LES smooth muscle membranes by increasing concentrations of unlabeled
VIP in absence (control) and presence of ZnPP IX and ZnDP IX (1 × 104 M). Note significant
shift in binding displacement curve in presence of ZnPP IX
(* P < 0.05, n = 6), whereas ZnDP IX had no
significant effect.
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DISCUSSION |
Multiple actions of ZnPP IX are well recognized. In the LES the
protoporphyrin caused blockade of the smooth muscle AC and guanylate
cyclase (GC) stimulation by VIP and atrial natriuretic factor or
peptide, respectively (14, 29). Although the nonspecificity of actions
of ZnPP IX may be widespread, in the present study we focused primarily
on the mechanism of its inhibitory action on VIP.
The studies show that ZnPP IX caused significant attenuation of the G
protein-coupled receptor activation by VIP. It is well known that the
major part of the relaxant action of VIP in the LES is mediated by
direct action at the VIP receptor on the smooth muscle cells via the
activation of AC (1, 9, 32, 36). To determine the site of action of
ZnPP IX in blocking VIP-induced relaxation of the LES smooth muscle, we
systematically investigated the influence of ZnPP IX on agents that
work along different steps to produce VIP receptor-mediated smooth
muscle relaxation. This included the examination of the actions of VIP
on the basal LES tone, VIP receptor binding, and comparison of the
actions of VIP vs. CTX before and after ZnPP IX.
CTX is known to cause direct activation of
Gs protein associated with AC (5,
41). The present studies in the LES showed for the first time that CTX
causes a concentration-dependent fall in the basal tension by its
action directly at the smooth muscle cells since it was not modified by
neuronal blockade. The convergence of VIP and CTX on AC activation to
produce LES relaxation was verified by their similar attenuation by the
AC inhibitor NEM. The convergence of the actions of VIP and CTX on a
similar intracellular pathway was further confirmed by the blockade of
the inhibitory action of VIP on the LES smooth muscle by CTX desensitization.
The data suggest that the fall in the basal tone in the LES induced by
VIP and CTX follows the same final biochemical pathway, i.e., the
stimulation of AC. Along this final pathway, however, the original
sites of action of VIP and CTX are different in causing the LES smooth
muscle relaxation. The action of VIP is mediated via activation of G
protein-coupled receptors and that of CTX is downstream, bypassing the
receptor interaction, and being exerted directly at the level of G
protein activation. This difference in the locus of action of these
agents in the present study played an important role in the
determination of the site of action of ZnPP IX in blocking the action
of VIP in the LES.
The inhibitory action of isoproterenol in the LES especially in the
lower concentrations was also attenuated by ZnPP IX (13). The
suppressant effect of ZnPP IX on isoproterenol-induced smooth muscle
relaxation may involve its interaction with the 
-adrenoceptors. The
data show that ZnPP IX blocks the action of VIP but has no significant
effect on the fall in the LES tension caused by CTX. This suggests that
the site of action of ZnPP IX in inhibiting the action of VIP lies at a
point between the receptor interaction and G protein activation. The
VIP binding experiments further confirmed that the major mechanism of
action of ZnPP IX in inhibiting VIP action is due to inhibition of the
coupling of VIP with the receptor. In separate studies (14) we have
shown that ZnPP IX causes no significant modification of the LES
relaxation by forskolin, a direct stimulator of AC that bypasses G
protein activation.
As stated above, ZnPP IX exerts multiple actions in the smooth muscle.
However, the inhibitory action of ZnPP IX on the G protein-coupled
receptor activation leading to LES smooth muscle relaxation cannot be
explained simply on the basis of complete nonselectivity for the
following reasons. The actions of the muscarinic agonist bethanechol
that stimulate a specific G protein-coupled receptor (10, 12, 19),
causing an increase in the basal tone of the LES (17), and the fall in
the LES tone induced by CTX (a polypeptide), SNP, and forskolin were
not modified. Furthermore, in the LES, unlike the IAS, the NANC nerve
stimulation-induced relaxation of the sphincteric smooth muscle was
also not modified by the HO inhibitor. We suggest that in the LES the
predominant pathway for the NANC nerve-induced relaxation is NOS. The
predominance of the NOS pathway in the LES relaxation is evident from
the previously published data that show the NOS inhibitor nearly
abolishes the relaxation by NANC nerve stimulation (30, 40).
In light of the strong evidence in favor of the VIP as an inhibitory
neurotransmitter (3, 20), it is rather surprising that ZnPP IX caused
near obliteration of VIP response but had no effect on the NANC
relaxation. One of the plausible explanations may be NOS upregulation.
A leftward shift in the EFS-frequency response curve in the feline LES
in the presence of ZnPP IX plus L-NNA compared with
L-NNA alone (28) and release of
NO by ZnPP IX in the rabbit IAS (7) may lend support to this
speculation. This may have important pathophysiological implications in
the counterregulation between HO and NOS pathways and in protecting the
tissues against the deleterious effects of overproduction of NO. The
colocalization of HO and NOS, as recently demonstrated in brain neurons
(39) and in the feline LES (28), further suggests this possibility.
Despite multiple and nonselective actions of ZnPP IX in different
smooth muscles, in the IAS the interaction between VIP and HO was found
to be relatively defined since the actions of a closely related
peptide, PHI, were not modified by the HO inhibitor (31). The
concentrations of ZnPP IX used in the present studies were similar to
those found to be effective in inhibiting HO activity in the LES (28).
Furthermore, ZnDP IX, which is known to block HO activity with a
greater potency than ZnPP IX, had a limited effect on the VIP-induced
relaxation of the LES as well as on VIP receptor binding. The data
suggest a lack of correlation between the inhibition of HO activity by
the porphyrins and their ability to block the responses to VIP.
Because of the nonselectivity of action of ZnPP IX, the exact role of
the HO pathway in the LES relaxation by NANC nerve stimulation cannot
be determined at the present time. The direct action of CO on the
smooth muscle via direct activation of GC, the presence of basal HO
activity, its increase by NANC nerve stimulation, and inhibition by
ZnPP IX in certain gastrointestinal tissues (28, 31), the presence of
HO-2 immunoreactivity in the myenteric plexuses (6, 6, 28, 34); the
electrophysiological correlation between CO and NANC relaxation (15);
the colocalization of HO with NOS and VIP immunoreactivities (2, 28,
34); and the inhibition of NANC nerve-mediated relaxation by the
selective knock-out of the HO-2 gene in certain gastrointestinal
tissues (31, 37, 42) suggest participation of the HO pathway in some
way in the gastrointestinal motility.
In conclusion, ZnPP IX causes blockade of the action of VIP at the LES
smooth muscle membrane receptor that is G protein coupled to AC. The
site of action of ZnPP IX is above the level of activation of G protein
since the effects of direct G protein activation by CTX were not
modified by ZnPP IX. The failure of ZnPP IX to modify NANC nerve
stimulation-induced relaxation of the LES may be explained by the
possibility that the smooth muscle relaxation by the NO released on
NANC nerve-mediated stimulation uses a unique receptor activation that
bypasses the G protein coupling to GC. The data further suggest that
ZnPP IX, especially at high concentrations, may not be a specific HO
inhibitor and that there is a need for a more selective HO inhibitor.
The discovery of such an agent may facilitate investigations of the
role of the HO pathway in gastrointestinal motility.
| |
ACKNOWLEDGEMENTS |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-35385 and an institutional grant
from Thomas Jefferson University.
| |
FOOTNOTES |
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: S. Rattan, 901 College, Dept. of
Medicine, Div. of Gastroenterology and Hepatology, 1025 Walnut St.,
Philadelphia, PA 19107.
Received 16 July 1998; accepted in final form 7 October 1998.
| |
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