Neural regulation of in vitro giant contractions in the rat colon

Asensio Gonzalez and Sushil K. Sarna

Departments of Surgery and Physiology, Medical College of Wisconsin, Milwaukee 53266; and Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The rat middle colon spontaneously generates regularly occurring giant contractions (GCs) in vitro. We investigated the neurohumoral and intracellular regulation of these contractions in a standard muscle bath. cGMP content was measured in strips and single smooth muscle cells. The circular muscle strips generated spontaneous GCs. Their amplitude and frequency were significantly increased by tetrodotoxin (TTX), omega -conotoxin, Nomega -nitro-L-arginine (L-NNA), and the dopamine D1 receptor antagonist Sch-23390. The GCs were unaffected by hexamethonium, atropine, and antagonists of serotonergic (5-HT1-4), histaminergic (H1-2), and tachykininergic (NK1-2) receptors but enhanced by NK3 receptor antagonism. The guanylate cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ) also enhanced GCs to the same extent as TTX and L-NNA, and each of the three agents prevented the effects of the others. GCs were abolished by electrical field stimulation, S-nitroso-N-acetyl-penicillamine, and 8-bromo-cGMP. BAY-K-8644 and apamin enhanced the GCs, but they were abolished by D-600. Basal cGMP content in strips was decreased by TTX, L-NNA, or ODQ, but these treatments had no effect on cGMP content of enzymatically dissociated single smooth muscle cells. We conclude that spontaneous contractions in the rat colonic muscle strips are not generated by cholinergic, serotonergic, or histaminergic input. Constitutive release of nitric oxide from enteric neurons sustains cGMP synthesis in the colonic smooth muscle to suppress spontaneous in vitro GCs.

nonadrenergic, noncholinergic; enteric neurons; nitric oxide; guanosine 3',5'-cyclic monophosphate; dopamine; cholinergic; tachykinins; apamin; calcium channels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE IN VIVO COLONIC MOTOR activity in most species, including dogs, humans, and rats, is characterized by three distinct types of contractions: 1) rhythmic phasic contractions, 2) giant migrating contractions (GMCs), and 3) tone (18, 22, 38, 39). The GMCs are large-amplitude and long-duration contractions that migrate uninterruptedly over long distances and are associated with mass movements and defecation (23, 24, 41). The spontaneous GMCs occur once or twice a day in the human and the dog colon. In the in vivo rat colon, however, they occur regularly at a frequency of ~40/h (22). In this species, the GMCs migrate infrequently and over much shorter distances than in humans or dogs. Previous studies have shown that the GMCs are not regulated by slow waves and their smooth muscle signaling pathways are different from those of phasic contractions (37, 40).

The role of enteric neurons in the regulation of colonic GMCs is not well understood. One of the limitations has been that the GMCs are not known to occur in canine and human in vitro muscle strips. A recent report by Gonzalez and Sarna (12) indicates, however, that circular muscle strips prepared from the middle rat colon generate regular giant contractions (GCs) as well as phasic contractions. This in vitro finding provides an opportunity to investigate the enteric neural regulation of GCs in a muscle bath environment. The specific aims of this study were to determine whether 1) there is a constitutive release of neurotransmitters to stimulate or inhibit GCs and 2) there is a differential enteric neural regulation of concurrently occurring GCs and phasic contractions. Our results indicated that there is constitutive release of nitric oxide (NO) and dopamine that suppresses the GCs. Therefore, our third aim was to determine the cellular pathways for the inhibition of spontaneous GCs by NO.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recording of spontaneous contractile activity. Male Sprague-Dawley rats (300-350 g in weight) fed with a standard chow diet were used for the study. The animals were given 50 mg/kg pentobarbital sodium intraperitoneally. The middle colon (5 cm from the cecum and 7 cm from the pelvic brim) was removed and immediately placed in Krebs solution at 37°C preequilibrated with carbogen. Mucosa-free strips, 3 mm wide × 10 mm long, were cut along the circular muscle axis and mounted in a 3-ml muscle bath filled with Krebs at 37°C and bubbled with carbogen. The tissues were left to equilibrate for at least 60 min, and the bath solution was changed every 15-20 min until GC frequency and amplitude stabilized. Both phasic contractions and GCs were observed at low levels of stretch. However, preliminary experiments were performed by stepwise stretching and measuring the contraction to 2 µM ACh. Optimal stretching was found at ~8-10 mN passive tension, and for subsequent experiments the strips were routinely stretched to 10 mN tension.

Pharmacological antagonists and neurotoxins were added to the muscle bath in volumes of <1% of the bath volume, and the effects were recorded for 10 min unless stated otherwise. The frequency was measured over the 10-min period before addition of drugs and during the 10 min after the addition, unless noted otherwise. In experiments with combined addition of antagonists and agonists, the antagonist was allowed to act for 10 min before adding the agonist. The contractions were recorded on a Grass 7D polygraph and analyzed by a digital data acquisition system (DATAQ Instruments, Akron, OH).

Preparation of single smooth muscle cells. The muscularis externa from the middle colon was dissected free of mucosa and submucosa with fine tweezers under a dissecting microscope. The tissue was incubated at 37°C in digestion solution containing 25 mM HEPES and 0.4 mg/ml papain (Sigma Chemical, St. Louis, MO) for 20-30 min and then in collagenase Worthington type II (0.2 mg/ml) until cells were released by pipetting of the digested tissue as assessed microscopically (30-60 min). The tissue was further pipetted with a silicone-coated Pasteur pipette, and the cells were washed twice with sterile DMEM (GIBCO, Rockville, MD) and incubated for 4 h in DMEM at 37°C in an atmosphere of 10% CO2 to allow the cells to recover from the digestion procedure. For cGMP measurement, the cells were lysed following the protocol described in the enzyme immunoassay kit (Amersham Life Science, Chicago, IL). The cell dispersions were filtered through a 200-µm nylon mesh, and the suspensions were checked with a phase contrast microscope to confirm the presence of smooth muscle cells in the absence of enteric ganglia, which were much larger in size.

Measurement of cGMP. The mechanical effect of pharmacological agents on muscle strips was checked before measuring their cGMP content. Then the strips were dried by blotting immediately (<10 s), frozen in liquid nitrogen, and stored at -80°C until assay. For analysis, the frozen tissue pieces were quickly weighed and homogenized in 6% trichloroacetic acid, the debris was pelleted by centrifugation, and the supernatants were washed three times with water-saturated ethyl ether to extract residual trichloroacetic acid. The extracts were lyophilized and reconstituted in 1 M potassium phosphate buffer, pH 7.4 (37). cGMP was determined by enzyme immunoassay (Amersham Life Sciences) following the acetylation protocol, with a lower limit of detection in the femtomolar range.

Analysis of data. Comparisons between two groups were performed by parametric Student's test; P < 0.05 was considered statistically significant. The n values represent the number of strips tested, and they were taken from at least three different animals. For biochemical measurements, standard curves were generated by nonlinear regression analysis of the experimental data points, and goodness of fit (r2) was always >0.99. All data are expressed as means ± SE.

Drugs. Tetrodotoxin (TTX), omega -conotoxin GVIA (omega -CTX), Nomega -nitro-L-arginine (L-NNA), MDL-12330A, apamin, 8-bromo-cGMP (8-BrcGMP), D-600 phentolamine, domperidone, mepyramine, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ), vasoactive intestinal peptide (VIP), VIP(10---28), and ranitidine were purchased from Sigma Chemical. L-703606, L-659977, Trp7,beta -Ala8-NKA4-10, suramin, pyridoxal phosphate-6-azophenyl-2',4'-disulfonoic acid (PPADS), reactive blue, S-nitroso-N-acetyl-penicillamine (SNAP), BAY-K-8644, NAN-190 HBr, LY-53857, SDZ-205557, and tropisetron were obtained from RBI-Sigma. KT-5823 was purchased from Calbiochem (San Diego, CA), atropine from Elkins-Sinn (Cherry Hill, NJ), and Sch-23390 from Schering-Plough (Atlanta, GA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The circular muscle strips from the rat middle colon generated regular phasic contractions and GCs (Fig. 1). The amplitude of phasic contractions occurring in between GCs was nearly uniform. In addition, there was typically a burst of two to six phasic contractions that occurred at the tail end of GCs and had a larger amplitude than of those that occurred in between GCs (Fig. 1).


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Fig. 1.   Enhancement of spontaneous giant contractions (GCs) in rat colonic circular muscle strips by omega -conotoxin GVIA (omega -CTX, 0.5 µM), tetrodotoxin (TTX, 1 µM), and Nomega -nitro-L-arginine (L-NNA, 0.1 mM).

Neural regulation of spontaneous contractions. The spontaneous phasic contractions occurred at a frequency of 7.1 ± 0.5/min with a mean amplitude of 6 ± 1 mN and GCs at a frequency of 0.21 ± 0.02/min with a mean amplitude of 30 ± 6 mN (n = 47). The blockade of fast sodium channel enteric neural conduction by 1 µM TTX or N-type Ca2+ channels by 0.5 nM omega -CTX immediately increased the frequency of GCs to 0.78 ± 0.05/min and 0.65 ± 0.05/min, respectively (Figs. 1 and 2), and amplitude to 61 ± 1 mN and 57 ± 1 mN, respectively (n = 5, P < 0.01 both). The amplitude and frequency of phasic contractions were unaltered except for those that occurred at the tail end of the GCs. Their amplitude was increased in 65% of the preparations by 47 ± 12% (n = 5, Fig. 1).


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Fig. 2.   Effects of different neural blockers, receptor antagonists, second messengers, and ion channel blockers on the frequency (A) and amplitude (B) of GCs in the circular muscle of the rat middle colon (P < 0.05 vs. control). ODQ, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one.

L-NNA (0.1 mM) also increased the GC frequency and amplitude (Fig. 1) to 0.79 ± 0.04/min and 60 ± 1 mN, respectively (P < 0.01, n = 5, Fig. 2). These values were not significantly different from those after neural or N-type Ca2+ channel blockade (P > 0.05). The blockade of NO synthesis also increased the amplitude of phasic contractions in between GCs by 68 ± 17% in 81% of the strips (n = 5).

Table 1 lists the pharmacological antagonists and their concentrations used to investigate the role of spontaneous neurotransmitter release in the genesis or modulation of GCs, phasic contractions, and tone. Atropine (1 µM), a nonspecific muscarinic receptor antagonist, reduced the GC frequency transiently, but it recovered in 10 min (Fig. 3). Atropine had no transient or long-term effect on spontaneous phasic contractions. The concentration of 1 µM atropine totally blocked the excitatory effect of 30 µM ACh, confirming that this dose was effective in blocking muscarinic receptors (Fig. 3).

                              
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Table 1.   Neurotransmitter receptor antagonists used in this study



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Fig. 3.   A: lack of antagonism of spontaneous rat colonic GCs by 1 µM atropine. ACh (30 µM) was added in the presence of atropine to illustrate that muscarinic blockade was complete. B: typical response of a different colonic strip to 30 µM ACh without atropine.

Hexamethonium, a nicotinic-receptor antagonist, and antagonists for adrenergic, serotonergic, histaminergic, VIP, pituitary adenylate cyclase-activating polypeptide (PACAP), and tachykininergic receptors did not affect the frequency or amplitude of GCs and phasic contractions with the exception of selective NK3-receptor antagonist Trp7,beta -Ala8-NKA4-10, which elevated the tone of muscle strips and increased the frequency of GCs to 0.51 ± 0.05/min (Fig. 4). This effect was resistant to 1 µM TTX, indicating that NK3 tachykininergic regulation of tone and GCs is nonneural in origin.


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Fig. 4.   A: effect of NK3-receptor antagonist Trp7, beta -Ala8-NKA4-10 (10 µM) on spontaneous contractions of the rat colonic circular muscle strips. The amplitude of the phasic contractions was suppressed, whereas GC frequency and tone were increased. B: dopamine D1-receptor antagonist Sch-23390 (10 µM) increased the frequency and amplitude of spontaneous GCs.

The nonspecific purinergic antagonist suramin did not alter the GC frequency or amplitude in the 10-min period after its addition to the bath. However, it increased the amplitude of the GCs to 41 ± 6 mN (n = 8, P < 0.05) 30 min after being added to the bath (Fig. 2). The frequency of GCs was not altered at this time. The role of purinergic receptors was examined further with selective antagonists. PPADS (P2X receptor antagonist) did not affect the spontaneous phasic contractions or GCs. Reactive blue (P2Y receptor antagonist) actually decreased the amplitude of GCs to 13 ± 1.3 mN (n = 4, P < 0.05). By contrast, dopamine D1 receptor antagonist Sch-233990 (10 µM) consistently increased the frequency to 0.71 ± 0.05/min and amplitude to 51 ± 12 mN of GCs (n = 7, P < 0.05, Fig. 2B and Fig. 4B). The selective D2 receptor antagonist domperidone (10 µM) caused an increase in GC amplitude to 39 ± 5 mN (n = 6, P < 0.05) without affecting the frequency. These antagonists had no effect on phasic contractions.

Intracellular regulation of GCs and phasic contractions. When the activation of soluble guanylate cyclase was blocked by 1 µM ODQ, the GC frequency and amplitude exhibited a similar immediate increase to 0.73 ± 0.06/min and 63 ± 2 mN (n = 9, P < 0.01) as was seen after L-NNA, TTX, and omega -CTX. The amplitude of phasic contractions in between the GCs was not affected, but that of contractions superimposed on the trailing edge of GCs was increased by 32 ± 5% in 45% of the strips (Fig. 5A). In contrast, the inhibition of adenylate cyclase by 10 µM MDL-12330 had no effect on the frequency or amplitude of GCs or phasic contractions 10 min after its addition to the muscle bath (Fig. 5B).


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Fig. 5.   A: enhancement of rat colonic spontaneous GCs by guanylate cyclase inhibitor ODQ (1 µM). ODQ increased both the frequency and amplitude of spontaneous GCs. B: by contrast, adenylate cyclase inhibitor MDL-12330A was without effect.

To elucidate whether TTX, L-NNA, and ODQ act through common pathways, the effect of each of these agents was examined in the presence of the others. Neither L-NNA nor ODQ further altered the frequency and amplitude of GCs after neural blockade with TTX (Fig. 6). Similarly, TTX had no effect on GCs in the presence of L-NNA or ODQ (Fig. 6). Furthermore, electrical field stimulation (EFS) at 10 Hz, 0.4 ms duration, 150 V for 5 min or the NO donor SNAP (100 µM) in the presence of atropine, phentolamine, and propranolol (1 µM each) completely abolished the GCs (Fig. 7, A and B). The phasic contractions persisted in the presence of EFS, but they were abolished by SNAP. The GCs were abolished by the membrane-permeable cGMP analog 8-BrcGMP (10 µM, Fig. 7C). In the presence of 10 µM 8-BrcGMP, neither TTX, nor L-NNA, nor ODQ were able to revive the GCs. As noted earlier, all three agents increased the frequency and amplitude of GCs without 8-BrcGMP.


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Fig. 6.   A and B: presence of TTX (1 µM) prevented L-NNA (0.1 mM) or ODQ (1 µM) from further increasing the frequency of GCs. C and D: likewise, ODQ (1 µM) or L-NNA (0.1 mM) prevented further enhancement of GCs by TTX (1 µM).



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Fig. 7.   Blockade of GCs by electrical field stimulation (A, EFS, 10 Hz, 0.4-ms duration, 150 V for 5 min), nitric oxide donor S-nitroso-N-acetyl-penicillamine (B, SNAP, 0.1 mM), and the permeable cGMP analog 8-bromo-cGMP (8-BrcGMP, C).

The inhibition of cGMP-dependent kinase (protein kinase G, PKG) with KT-5823 (1 µM) had no effect on GCs, indicating that cGMP inhibits GCs independent of the activation of PKG.

An alternate pathway for the inhibition of cell contractions by cGMP involves the K+ channels (21, 27). The blockade of Ca2+-activated K+ channels with 1 µM apamin increased the frequency of GCs to 0.63 ± 0.04/min and amplitude to 69 ± 9 mN (n = 4, P < 0.01, Fig. 8C). In the presence of TTX or L-NNA, apamin did not further affect the frequency, but it further increased the GC amplitude to 87 ± 10 mN (n = 3, P < 0.05). In the presence of apamin, ODQ further increased the GC frequency to 0.98 ± 0.04/min (n = 3, P < 0.05).


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Fig. 8.   A: enhancement of GCs in the rat colon by BAY-K-8644 (1 µM). B: blockade by L-type calcium channel blocker D-600 (1 µM) of all mechanical activity. C: increase by Ca2+-activated small-conductance K+ channel blocker apamin (1 µM) of the frequency and amplitude of GCs. D: lack of effect of the nonspecific blocker of cation channels gadolinium on spontaneous GCs.

The Ca2+ channel opener BAY-K-8644 (1 µM) increased the GC frequency to 0.76 ± 0.07/min and amplitude to 67 ± 3 mN (P < 0.05, n = 5). All GCs and phasic contractions were totally blocked by 1 µM D-600 (Fig. 8B). Thus Ca2+ influx through L-type channels is necessary for both phasic contractions and GCs. In contrast, gadolinium chloride (10 µM), a nonspecific blocker of cation channels (20, 29) had no effect on the GCs or phasic contractions.

The origin of NO inducing cGMP synthesis. The cGMP content in the muscularis externa containing the myenteric plexus was 39 ± 3 pmol/g tissue weight. When the muscle strips were incubated with 1 µM TTX, 0.1 mM L-NNA, or 1 µM ODQ, the cGMP content significantly decreased to 25 ± 3, 19 ± 2, and 22 ± 3 pmol/g tissue weight, respectively (n = 5, P < 0.05 for each, Fig. 9A).


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Fig. 9.   cGMP synthesis in rat colonic muscle strips (A) and dispersed smooth muscle cells (B) in response to 1 µM TTX, 0.1 mM L-NNA, and 1 µM ODQ. In B, the cells treated with 10 µM SNAP increased their cGMP content by 86%.

We then measured cGMP in dispersed smooth muscle cells devoid of enteric ganglia. The cGMP content in dispersed cells was 0.24 ± 0.05/106 cells. When the cells were incubated with 1 µM TTX, 0.1 mM L-NNA, or 1 µM ODQ, the cGMP content was 0.22 ± 0.07, 0.23 ± 0.02, and 0.19 ± 0.04/106 cells, respectively (n = 5, P > 0.05 for each, Fig. 9B). We checked whether the dispersed cells retained their ability to synthesize cGMP in response to NO. An aliquot of dispersed cells was incubated for 10 min with the NO donor 0.1 mM SNAP; the cGMP level increased to 0.41 ± 0.06/106 cells (n = 4, P < 0.05, Fig. 9B), thus confirming that guanylate cyclase activation by NO is maintained in the dispersed cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inhibitory neural regulation of GCs. We (12) and Middleton et al. (25) have noted that circular muscle strips prepared from the rat middle colon generate spontaneous GCs as well as rhythmic phasic contractions. The generation of spontaneous phasic contractions by muscle strips is well established, but the generation of spontaneous and regularly occurring GCs was novel. We therefore used these strips to investigate the enteric neural regulation of GCs. Our findings show that in the rat colon, the GCs are tonically inhibited by nonadrenergic, noncholinergic (NANC) inhibitory neurons. The inhibition of fast Na+ channel enteric neural conduction by TTX or the blockade of N-type neural Ca2+ channels by omega -CTX increased the frequency and amplitude of GCs.

Anuras and Christensen (2) reported tonic inhibition of in vitro rhythmic phasic contractions by NANC neurons in cat colonic circular muscle strips. They found that TTX enhanced the amplitude of spontaneous contractions of colonic circular but not longitudinal muscle strips in this species (2). Wood (49) found that TTX enhanced the spontaneous phasic contractions in isolated segments of the cat jejunum. By contrast, Rae et al. (34) found that TTX had no effect on the spontaneous contractions of the human colonic circular muscle strips, whereas it enhanced the amplitude of phasic contractions in the canine colon (19). In rat middle colon, TTX had no significant effect on phasic contractions, but at the same time it enhanced the GCs. TTX did increase the amplitude of phasic contractions that occurred on the falling phase of the GCs. However, the basal conditions for these phasic contractions may be different from those that occur in between GCs. For example, the membrane is likely to be depolarized during a GC (37). Interestingly, the phasic contractions persisted in the presence of EFS at 10 Hz, 0.4 ms, 150 V for 5 min, even though it blocked the GCs. On the other hand, SNAP, an NO donor, and 8-BrcGMP both blocked the phasic contractions as well as GCs. The differences between the effects of TTX and EFS on spontaneous phasic contractions and GCs is likely to be due to a difference in the thresholds of inhibitory effects on the two types of contractions. The ongoing spontaneous inhibitory input or that on EFS is enough to suppress the GCs but not the phasic contractions. The GCs are large-amplitude and long-duration contractions and therefore they may maximally utilize the cellular resources such as cytosolic Ca2+ to contract, hence their reduced susceptibility to inhibitory input.

In muscle strips, it seems that the spontaneous generation of GCs is not dependent on the release of a neurotransmitter from the known excitatory cholinergic, serotoninergic, histaminergic, or tachykininergic neurons. The antagonists of their receptors (or receptor subtypes) had no effect on the frequency or amplitude of GCs. By contrast, Li et al. (22) found that in the in vivo intact conscious state, the spontaneous GCs in the rat colon are blocked by atropine. In muscle bath, atropine transiently reduced the frequency of GCs, but it recovered within 10 min. One possibility is that stretch, which is an essential feature of muscle bath and induces Ca2+ influx, is able to trigger the same cellular mechanisms in vitro that ACh does in vivo to stimulate GCs. No spontaneous rhythmic contractions were observed visually in strips when they were not stretched. Other reports have indicated that stretch opens nonselective cation channels in smooth muscle cells (16). We found, however, that gadolinium chloride, a blocker of nonspecific cation channels, did not block the GCs or phasic contractions. On the other hand, the blockade of L-type Ca2+ channels totally inhibited all spontaneous contractions. Farrugia et al. (10) have reported stretch-activated L-type Ca2+ channels in human intestinal smooth muscle cells. The stretch on muscle strips therefore may open the L-type channels to activate the cellular processes that produce these contractions. This hypothesis is supported by the observation that the opening of L-type channels by BAY-K-8640 increased the frequency and amplitude of GCs.

The inhibition of NK3 receptor increased the basal tone of muscle strips as well as the frequency of GCs. This was a direct effect of NK3 antagonist on smooth muscle cells because it was not blocked by TTX. Of all the antagonists used in this study, NK3 antagonist was the only one that increased muscle tone. The others selectively affected the frequency and amplitude of GCs or had no effect. This confirms other reports that the cellular processes for the generation of different types of contractions may differ (40).

Inhibitory neurotransmitters. Further examination of the tonic inhibitory input to suppress GCs indicated that both NO and dopamine may be involved in this phenomenon. The inhibition of NO synthesis immediately increased the amplitude and frequency of GCs to the same extent as that by TTX or omega -CTX. Middleton et al. (25) also found that a constitutive release of NO enhances the frequency of what they called summation contractions in the rat distal colon. We (12) showed, however, that the GCs are distinct contractions and that they are not due to the summation of phasic contractions. The inhibition of D1 receptors by Sch-233990 had a similar effect. On the other hand, the blockade of D2 receptors by domperidone increased only the amplitude of GCs without affecting their frequency. The reasons for this difference in the response between the blockade of D1 and D2 receptors are not known, but it is likely that each receptor is coupled to a different signal transduction pathway to produce its inhibitory effect. By contrast, in the dog and the human colon dopamine has been found to be excitatory rather than inhibitory (5, 48).

The addition of ODQ, Sch-233990, or domperidone to the muscle bath enhanced the GCs, but it had no significant effect on the phasic contractions. Franck et al. (11) also found that ODQ has no effect on spontaneous phasic contractions in the canine proximal colon. These observations highlight the differences in the regulation of spontaneous GCs and phasic contractions by tonic inhibitory input. The exception to the above observations is that nitro-L-arginine methyl ester (L-NAME) increased the frequency of phasic contractions in ~65% of the muscle strips. The precise reasons for this anomaly are not known.

In contrast to NO and dopamine antagonists, the antagonism of VIP receptors by VIP(10---28) and purinergic receptors by suramin, PPADS (P2x), and reactive blue (P2y) did not increase the frequency of GCs. The inhibition of adenylate cyclase by MDL-12330 also had no effect on spontaneous GCs. It therefore seems that ATP and VIP-PACAP are not released spontaneously by the NANC inhibitory neurons to inhibit GCs. Plujà et al. (32) found, however, that suramin increases the amplitude and frequency of phasic contractions in the rat colon. They did not specify the part of the colon used in their study. It is possible that the expression of ATP may be different in different parts of the colon, as is the case with NO (43).

Cellular mechanisms of NO-mediated inhibition of GCs. Previous studies have reported that NO activates guanylate cyclase to inhibit cell contractility (7, 13, 45, 47). Our findings show that this cellular pathway is utilized by the spontaneous release of NO to inhibit GCs. ODQ that inhibits the activation of guanylate cyclase increased the frequency and amplitude of GCs similar to that seen after TTX, omega -CTX, and L-NNA. In addition, 8-BrcGMP inhibited the GCs.

cGMP has been reported to inhibit smooth muscle contractility by the activation of PKG and by the opening of Ca2+-dependent small conductance K+ channels (35). In our study, the inhibition of PKG by KT-5823 did not affect the spontaneous GCs. Smaller concentrations of KT-5823 have been shown to inhibit the activation of PKG in other cell types (44). On the other hand, the inhibition of Ca2+-dependent small- conductance K+ channels by apamin increased the frequency and amplitude of GCs to the same extent as that seen after TTX, L-NNA, or ODQ. There may, however, be an additional pathway, such as the regulation of the sensitivity of the contractile proteins to Ca2+ (29) for the inhibition of GCs by cGMP because in the presence of apamin, ODQ further increased the frequency of GCs. This pathway is undefined.

The spontaneous release of NO and subsequent formation of cGMP is a constitutive phenomenon independent of stretch. In unstretched muscle strips TTX, L-NNA, or ODQ reduced the basal levels of cGMP in tissues containing longitudinal muscle, circular muscle, and the myenteric plexus. By contrast, these agents had no effect on the basal cGMP content of dispersed circular smooth muscle, even though these cells maintained their ability to synthesize cGMP in response to NO. These findings confirmed that NO was being released from the enteric neurons.

Physiological and clinical relevance. Most previous studies (3, 4, 6, 9, 28, 31, 42) have investigated the inhibitory role of NO in gut motility by monitoring the relaxation of stretch or agonist-induced tone in muscle strips or by monitoring inhibitory junctional potentials. Whereas a physiological role of tone is well defined in sphincteric muscle (1, 36, 46), its physiological role in the main organs of the gastrointestinal tract is not fully understood. For this reason, we focused our studies on the potential role of NO in the generation of spontaneous phasic contractions and GCs whose motility roles in mixing and propulsion of digesta are well defined (8, 17, 33, 41). The phasic contractions cause the orderly slow distal propulsion of luminal contents in the fasting and postprandial states, whereas GMCs cause mass movements. Our findings show that a constitutive release of NO and dopamine may suppress the amplitude and frequency of spontaneous in vitro GCs in the rat middle colon.

Mizuta et al. (26) found that the inhibition of NO synthesis by L-NAME in intact rats retards colonic transit. Studies in our laboratory have shown that L-NAME increases the frequency of GCs in intact rats (22), which is similar to that seen in muscle strips. Mizuta et al. (26) proposed that the slower transit after L-NAME may be due to the impairment of descending relaxation. Our findings suggest that the delayed transit after the inhibition of NO synthase may also be due to the uncontrolled stimulation of spontaneous GCs in this species. The suppression of GCs in the untreated colon may produce spatial coordination so that they are more effective in propulsion. If so, the inhibitory neurons may have an important role in producing not only descending inhibition (14, 15) but also partially inhibiting the spontaneous contractions to facilitate propulsion. Orihata and Sarna (30) also found that the inhibition of NO synthase increases the frequency of pyloric and duodenal contractions without a concurrent coordination of contractions across the pylorus and therefore delays gastric emptying. The degree of this nitrergic inhibition may, however, vary among different species because available evidence indicates that the neural inhibition of spontaneous contractions in dog and human colon may be less pronounced than that in the rat colon.

We conclude that a constitutive release of NO and dopamine inhibit the GCs but not the phasic contractions in the rat mid-colon. The NO-induced inhibition is mediated by cGMP and Ca2+-dependent small conductance K+ channels. PKG does not seem to be involved. These findings, together with those observed in vivo (26), suggest that this inhibition may be important in normal transit in the rat colon.


    ACKNOWLEDGEMENTS

This work was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32346 and the Department of Veterans Affairs Medical Research Service.


    FOOTNOTES

Address for reprint requests and other correspondence: S. K. Sarna, General Surgery, Medical College of Wisconsin-FWC, 9200 West Wisconsin Ave., Milwaukee, WI 53226 (E-mail: ssarna{at}mcw.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 28 August 2000; accepted in final form 7 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allescher, HD, Tougas G, Vergara P, Lu S, and Daniel EE. Nitric oxide as a putative nonadrenergic noncholinergic inhibitory transmitter in the canine pylorus in vivo. Am J Physiol Gastrointest Liver Physiol 262: G695-G702, 1992[Abstract/Free Full Text].

2.   Anuras, S, and Christensen J. Effects of autonomic drugs on cat colonic muscle. Am J Physiol Gastrointest Liver Physiol 240: G361-G364, 1981[Abstract/Free Full Text].

3.   Boeckxstaens, GE, Pelckmans PA, Bult H, de Man JG, Herman AG, and van Maercke YM. Evidence for nitric oxide as mediator of non-adrenergic, non-cholinergic relaxations induced by ATP and GABA in the canine gut. Br J Pharmacol 102: 434-438, 1991[Abstract].

4.   Boeckxstaens, GE, Pelckmans PA, Herman AG, and van Maercke YM. Involvement of nitric oxide in the inhibitory innervation of the human isolated colon. Gastroenterology 104: 690-697, 1993[ISI][Medline].

5.   Bueno, L, Fargeas MJ, Fioramonti J, and Honde C. Effects of dopamine and bromocriptine on colonic motility in dog. Br J Pharmacol 82: 35-42, 1984[Abstract].

6.   Burleigh, DE. N G-nitro-L-arginine reduces nonadrenergic, noncholinergic relaxations of human gut. Gastroenterology 102: 679-683, 1992[ISI][Medline].

7.   Conklin, JL, and Du C. Guanylate cyclase inhibitors: effect on inhibitory junction potentials in esophageal smooth muscle. Am J Physiol Gastrointest Liver Physiol 263: G87-G90, 1992[Abstract/Free Full Text].

8.   Cowles, VE, and Sarna SK. Relation between small intestinal motor activity and transit in secretory diarrhea. Am J Physiol Gastrointest Liver Physiol 259: G420-G429, 1990[Abstract/Free Full Text].

9.   Dalziel, HH, Thornbury KD, Ward SM, and Sanders KM. Involvement of nitric oxide synthetic pathway in inhibitory junction potentials in canine proximal colon. Am J Physiol Gastrointest Liver Physiol 260: G789-G792, 1991[Abstract/Free Full Text].

10.   Farrugia, G, Holm AN, Rich A, Sarr MG, Szurszewski JH, and Rae JL. A mechanosensitive calcium channel in human intestinal smooth muscle cells. Gastroenterology 117: 900-905, 1999[ISI][Medline].

11.   Franck, H, Sweeney KM, Sanders KM, and Shuttleworth CW. Effects of a novel guanylate cyclase inhibitor on nitric oxide-dependent inhibitory neurotransmission in canine proximal colon. Br J Pharmacol 122: 1223-1229, 1997[Abstract].

12.   Gonzalez, A, and Sarna SK. Different types of contractions in rat colon and their modulation by oxidative stress. Am J Physiol Gastrointest Liver Physiol 280: G546-G554, 2001[Abstract/Free Full Text].

13.   Goyal, RK, and Xue DH. Evidence for NO · redox form of nitric oxide as nitrergic inhibitory neurotransmitter in gut. Am J Physiol Gastrointest Liver Physiol 275: G1185-G1192, 1998[Abstract/Free Full Text].

14.   Grider, JR. Interplay of VIP and nitric acid in regulation of the descending relaxation phase of peristalsis. Am J Physiol Gastrointest Liver Physiol 264: G334-G340, 1993[Abstract/Free Full Text].

15.   Hata, F, Ishii T, Kanada A, Yamano N, Kataoka T, Takeuchi T, and Yagasaki O. Essential role of nitric oxide in descending inhibition in the rat proximal colon. Biochem Biophys Res Commun 172: 1400-1406, 1990[ISI][Medline].

16.   Hisada, T, Walsh JV, Jr, and Singer JJ. Stretch-inactivated cationic channels in single smooth muscle cells. Pflügers Arch 422: 393-396, 1993[ISI][Medline].

17.   Holdstock, DJ, Misiewicz JJ, Smith T, and Rowlands EN. Propulsion (mass movements) in the human colon and its relationship to meals and somatic activity. Gut 11: 91-99, 1970[ISI][Medline].

18.   Karaus, M, and Sarna SK. Giant migrating contractions during defecation in the dog colon. Gastroenterology 92: 925-933, 1987[ISI][Medline].

19.   Keef, KD, Murray DC, Sanders KM, and Smith TK. Basal release of nitric oxide induces an oscillatory motor pattern in canine colon. J Physiol (Lond) 499: 773-786, 1997[Abstract].

20.   Kirber, M, Guerrero-Hernandez A, Bowman DS, Fogarty KE, Tuft RA, Singer JJ, and Fay FS. Multiple pathways responsible for the stretch-induced increase in Ca2+ concentration in toad stomach smooth muscle cells. J Physiol (Lond) 524: 3-17, 2000[Abstract/Free Full Text].

21.   Koh, SD, Campbell JD, Carl A, and Sanders KM. Nitric oxide activates multiple channels in canine colonic smooth muscle. J Physiol (Lond) 489: 735-743, 1995[Abstract].

22.   Li, M, Johnson CP, Adams MB, and Sarna SK. Ileal and colonic motor activity in dextran sodium sulfate-induced colitis in conscious rats (Abstract). Gastroenterology 116: G4470, 1999.

23.   Malcolm, A, and Camilleri M. Coloanal motor coordination in association with high-amplitude colonic contractions after pharmacological stimulation. Am J Gastroenterol 95: 715-719, 2000[ISI][Medline].

24.   Matsufuji, H, Yokoyama J, Hirabayashi T, Wantanabe S, and Sakurai K. Cooperative roles of colon and anorectum during spontaneous defecation in conscious dogs. Dig Dis Sci 43: 2042-2047, 1998[ISI][Medline].

25.   Middleton, SJ, Cuthbert AW, Shorthouse M, and Hunter JO. Nitric oxide affects mammalian distal colonic smooth muscle by tonic neural inhibition. Br J Pharmacol 108: 974-979, 1993[Abstract].

26.   Mizuta, Y, Takahashi T, and Owyang C. Nitrergic regulation of colonic transit in rats. Am J Physiol Gastrointest Liver Physiol 277: G275-G279, 1999[Abstract/Free Full Text].

27.   Mule, F, D'Angelo S, and Serio R. Tonic inhibitory action by nitric oxide on spontaneous mechanical activity in rat proximal colon: involvement of cyclic GMP and apamin-sensitive K+ channels. Br J Pharmacol 127: 514-520, 1999[Abstract/Free Full Text].

28.   Murray, J, Du C, Ledlow A, Bates JN, and Conklin JL. Nitric oxide: mediator of nonadrenergic noncholinergic responses of opossum esophageal muscle. Am J Physiol Gastrointest Liver Physiol 261: G401-G406, 1991[Abstract/Free Full Text].

29.   Nishimura, J, and van Breemen C. Direct regulation of smooth muscle contractile elements by second messengers. Biochem Biophys Res Commun 163: 929-935, 1989[ISI][Medline].

30.   Orihata, M, and Sarna SK. Inhibition of NO synthase delays gastric emptying of solid meals. J Pharmacol Exp Ther 271: 660-670, 1994[Abstract].

31.   Osthaus, LE, and Galligan JJ. Antagonists of nitric oxide synthesis inhibit nerve-mediated relaxations of longitudinal muscle in guinea pig ileum. J Pharmacol Exp Ther 260: 140-145, 1992[Abstract].

32.   Plujà, L, Fernández E, and Jiménez M. Neural modulation of the cyclic electrical and mechanical activity in the rat colonic circular muscle: putative role of ATP and NO. Br J Pharmacol 126: 883-892, 1999[Abstract/Free Full Text].

33.   Quigley, EMM, Phillips SF, and Dent J. Distinctive patterns of interdigestive motility of the canine ileocolonic junction. Gastroenterology 87: 836-844, 1984[ISI][Medline].

34.   Rae, MG, Fleming N, McGregor DB, Sanders KM, and Keef KD. Control of motility patterns in the human colonic circular muscle layer by pacemaker activity. J Physiol (Lond) 510: 309-320, 1998[Abstract/Free Full Text].

35.   Rapoport, RM, and Murad F. Agonist induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cyclic GMP. Circ Res 52: 352-357, 1983[Abstract].

36.   Rattan, S, and Chakder S. Role of nitric oxide as a mediator of internal anal sphincter relaxation. Am J Physiol Gastrointest Liver Physiol 262: G107-G112, 1992[Abstract/Free Full Text].

37.   Sarna, SK. Giant migrating contractions and their myoelectric correlates in the small intestine. Am J Physiol Gastrointest Liver Physiol 253: G697-G705, 1987[Abstract/Free Full Text].

38.   Sarna, SK. Physiology and pathophysiology of colonic motor activity (1). Dig Dis Sci 36: 827-862, 1991[ISI][Medline].

39.   Sarna, SK. Physiology and pathophysiology of colonic motor activity (2). Dig Dis Sci 36: 998-1018, 1991[ISI][Medline].

40.   Sarna, SK. Differential signal transduction pathways to stimulate colonic giant migrating and phasic contractions (Abstract). Gastroenterology 118: 4404, 2000.

41.   Sethi, AK, and Sarna SK. Contractile mechanisms of colonic propulsion. Am J Physiol Gastrointest Liver Physiol 268: G530-G538, 1995[Abstract/Free Full Text].

42.   Shuttleworth, CWR, Murphy R, and Furness JB. Evidence that nitric oxide participates in non-adrenergic inhibitory transmission to intestinal muscle in the guinea-pig. Neurosci Lett 130: 77-80, 1991[ISI][Medline].

43.   Takahashi, T, and Owyang C. Regional differences in the nitrergic innervation between the proximal and the distal colon in rats. Gastroenterology 115: 1504-1512, 1998[ISI][Medline].

44.   Tejedo, J, Bernabe JC, Ramirez R, Sobrino F, and Bedoya FJ. NO induces a cGMP-independent release of cytochrome C from mitochondria which precedes caspase 3 activation in insulin producing RINm5F cells. FEBS Lett 459: 238-243, 1999[ISI][Medline].

45.   Thornbury, KD, Ward SM, Dalziel HH, Carl A, Westfall DP, and Sanders KM. Nitric oxide mimics non-adrenergic, non-cholinergic hyperpolarization in gastrointestinal muscles. Am J Physiol Gastrointest Liver Physiol 261: G553-G557, 1991[Abstract/Free Full Text].

46.   Tottrup, A, Svane D, and Forman A. Nitric oxide mediating NANC inhibition in opossum lower esophageal sphincter. Am J Physiol Gastrointest Liver Physiol 260: G385-G389, 1991[Abstract/Free Full Text].

47.   Ward, SM, Dalziel HH, Bradley ME, Buxton ILO, Keef K, Westfall DP, and Sanders KM. Involvement of cyclic GMP in non-adrenergic, non-cholinergic inhibitory neurotransmission in dog proximal colon. Br J Pharmacol 107: 1075-1082, 1992[Abstract].

48.   Wiley, J, and Owyang C. Dopaminergic modulation of rectosigmoid motility: action of domperidone. J Pharmacol Exp Ther 242: 548-551, 1987[Abstract].

49.   Wood, JD. Excitation of intestinal muscle by atropine, tetrodotoxin, and xylocaine. Am J Physiol 222: 118-125, 1972[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 281(1):G275-G282