Introduction

Top Abstract Introduction Results Discussion References

Endothelins belong to a family of vasoconstrictor peptides that were initially thought to be associated with the regulation of cardiovascular function. More recently, endothelins have been suggested to play a broader role in diverse physiological actions (Rubanyi and Polokoff, 1994; Rae et al., 1995). Endothelins have been shown to be localized in the enteric nervous system (Takahashi et al., 1990; Inagaki et al., 1991) and have been proposed to exert important modulatory actions in the gastrointestinal motility (Takahashi et al., 1990; Allcock et al., 1995; Miasiro et al., 1995; Rae et al., 1995). Most of the actions of endothelins in the gastrointestinal tract are contractile and occur via their direct actions at the smooth muscle (Kitsukawa et al., 1994; Okabe et al., 1995). Furthermore, endothelins have been proposed to play a role in the development of the enteric nervous system (Baynash et al., 1994). It has been suggested that targeted disruption of the endothelin B (ET_B) receptor gene results in an aganglionosis of the colon that resembles Hirschsprung's disease in humans (Puffenberger et al., 1994). In our recent studies in an appropriate animal model, Ls/Ls mice, we have shown that Hirschsprung's disease is characterized by the loss of nitrergic inhibitory neurotransmission in the internal anal sphincter (IAS) (Chakder et al., 1997). In spite of their widespread distribution, the actions of endothelins on the gastrointestinal smooth muscle of the IAS are not known.

Two types of endothelin receptors, endothelin A (ET_A) and ET_B, have been identified based on their actions on the smooth muscle. In general, ET_B receptor activation is known to cause the smooth muscle relaxation; contraction, on the other hand, may either be via both ET_A and ET_B receptors or via ET_A receptor only (Kobari et al., 1994; Irie et al., 1995; Miasiro et al., 1995; Lucas et al., 1996; Higashi et al., 1997). The purpose of the present investigation was to examine the effects and mechanism of action of endothelins on the basal tone of the IAS.

Materials and Methods

Preparation of Smooth Muscle Strips. The IAS smooth muscle strips from opossums (Didelphis virginiana) were prepared for the recording of isometric tension as described previously (Rattan and Chakder, 1992). Briefly, following anesthesia with pentobarbital (40 mg/kg, i.p.), the animals were sacrificed by exsanguination and the anal canal along with a section of the rectum was isolated and transferred to oxygenated (95% O_2//5% CO₂) Krebs' solution of the following composition: NaCl, 118.07 mM; KCl, 4.69 mM; CaCl₂, 2.52 mM; MgSO₄, 1.16 mM; NaH₂PO₄ , 1.01 mM; NaHCO₃, 25 mM; and glucose, 11.10 mM. The anal canal 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 the IAS circular smooth muscle strips (1 × 10 mm) were prepared.

Measurement of Isometric Tension. The smooth muscle strips were tied at both ends with silk sutures (6-0; Ethicon Inc., 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 Co., 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 with 9.8 mN 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 (L_o) and the baseline of the smooth muscle strips were determined as explained before (Rattan and Chakder, 1992).

Nonadrenergic Noncholinergic (NANC) Nerve Stimulation with Electrical Field Stimulation (EFS). EFS was delivered from a Grass stimulator (model S88; Grass Instruments) connected in series to a Med-Lab Stimu-Splitter II (Med-Lab Instruments, Loveland, CO). The Stimusplitter served an important purpose to amplify and measure the actual stimulus intensity delivered to the tissues under the existing experimental conditions, 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. These parameters are known to cause the IAS smooth muscle relaxation via selective activation of NANC myenteric neurons (Rattan and Chakder, 1992; Chakder and Rattan, 1993a; Rattan et al., 1995). The electrodes used for the EFS consisted of a pair of platinum wires fixed at both sides of the smooth muscle strip.

Drugs and Chemicals. The following chemicals were used in the study: endothelin 1 and endothelin 2 (Bachem Bioscience Inc., King of Prussia, PA); atropine sulfate (muscarinic antagonist), guanethidine (adrenergic blocker), spantide (Substance P antagonist), tetrodotoxin (TTX; sodium-mediated axonal conduction blocker), indomethacin, L-N^G-nitro-arginine [nitric oxide synthase (NOS) inhibitor] and N-type neuronal Ca⁺⁺ channel blocker, -conotoxin GVIA (Sigma Chemical Co., St. Louis, MO); EDTA tetrasodium (Ca⁺⁺ chelator) (Fisher Scientific, Pittsburgh, PA); 1-(2-trifluoromethylphenyl) imidazole (TRIM; neuronal NOS inhibitor); 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7; protein kinase C inhibitor), and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13; calmodulin inhibitor) (Research Biochemicals International, Natick, MA).

5

Drug Responses. The effects of different concentrations of endothelin 1 and endothelin 2 were examined using single concentrations because of the tachyphylaxis problem when used cumulatively. The concentrations of different antagonists were maximally effective in blocking the actions of their respective agonists or enzymes and were relatively selective against the intended effects. Once the concentration-response curve to an endothelin was determined, the smooth muscle strips were washed 10 to 15 times over 2 h, and the resting tension was allowed to recover to the preinjection levels. Different antagonists or inhibitors except indomethacin were added 10 min before the addition of the agonists to the muscle bath. The smooth muscle strips were pretreated with indomethacin for 30 min before testing the effects of endothelin 1. Endothelin 1 tachyphylaxis was achieved by its frequent administration in the maximal effective concentration.

Data Analysis. The results are expressed as means and S.E. of different experiments. The fall of the resting IAS tension is expressed as the percentage of E_max (100%) in response to the supramaximal concentration (5 mM) of EDTA. The rise in tension is expressed as the percentage of E_max (100%) obtained with phenylephrine (1 × 10⁵ M). Statistical significance between different groups was determined by using paired or unpaired t test or analysis of variance where applicable, and a p value smaller than 0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Results Discussion References

Effect of Endothelin 1 on the Basal Tone of the IAS Smooth Muscle: Influence of Atropine and Guanethidine. In the initial experiments, we carried out concentration-response studies by the cumulative concentrations of endothelin 1. In these experiments, endothelin 1 was found to cause primarily concentration-dependent rise in the basal tension of the IAS (Fig. 1). However, in some of these experiments, there was an indication of the fall in the basal IAS tone, especially in the lower concentration range (1 × 10⁹- 1 × 10⁷ M). To further examine the divergent effects of endothelins in detail, subsequent studies were performed using single boluses. Under these experimental conditions, we observed a clear dichotomy of the inhibitory and excitatory effects of endothelin 1. There was clear evidence of a biphasic and concentration-dependent effect of endothelin 1 on the basal tone of the IAS. The biphasic effect consisted of an initial brief relaxation followed by a sustained contraction of the IAS smooth muscle. The initial fall was markedly more prone to tachyphylaxis than the contraction. Therefore, for the detailed pharmacological analyses of the concentration-response curves, extreme care was taken to wash the smooth muscle repeatedly to ensure the reversal of the control responses before pursuing the studies. Furthermore, a given smooth muscle was subjected to a limited experimental protocol. The actions of endothelin 2 were found to be similar to those of endothelin 1. 

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effect of endothelin 1 on the basal tone of the IAS smooth muscle. Endothelin 1 caused a biphasic effect in the IAS that consisted of an initial fall followed by a sustained rise in the basal tone of the IAS smooth muscle. The data in this and other figures show mean and S.E. The data in this figure represent mean and S.E. of five observations at each concentration level given in single boluses. The inset on the right is a typical example of the action of endothelin 1 (1 × 106 M) on the basal tone of the IAS smooth muscle.

6

6

6

6

Influence of Substance P Antagonist Spantide on the IAS Contraction Caused by Endothelin 1. Because the major action of Substance P in the IAS was to cause contraction of the IAS smooth muscle, only the contractile action of endothelin 1 was examined in the presence of spantide (3 × 10⁵ M). The data show that the rise in the IAS smooth muscle tension by endothelin was not significantly modified by spantide. The rise in the basal tension of the IAS in response to endothelin 1 (1 × 10⁶ M) before and after spantide were 54.8 ± 11.6 and 49.6 ± 4.4% respectively (p > .05; n = 4). The concentration of spantide used was found to be maximally effective in antagonizing the actions of Substance P in the IAS smooth muscle.

Influence of TTX on the Actions of Endothelin 1. The neurotoxin TTX, in the concentration (1 × 10⁶ M) that abolishes the EFS-induced relaxation of the IAS, failed to modify both the relaxant and the contractile actions of endothelin 1 on the basal tone of the IAS smooth muscle.

Influence of the Cycloxygenase Inhibitor Indomethacin on the Actions of Endothelin 1. Pretreatment with indomethacin (1 × 10⁵ M) for 30 min had no significant effect on either the fall or the rise in the basal IAS tension caused by endothelin 1. In these experiments, the initial falls in the basal tone of the IAS with 1 × 10⁷ M and 1 × 10⁶ M endothelin 1 were 26.6. ± 6.9 and 38.9 ± 4.0%, and the rises were 33.4 ± 8.9 and 47.4 ± 6.0%, respectively. These values for relaxation following indomethacin pretreatment were 25.9 ± 6.4 and 38.9 ± 4.0%, and for contraction were 50.0 ± 7.0 and 60.6 ± 7.6%, respectively (p > .05; n = 5). Furthermore, indomethacin pretreatment failed to influence the tachyphylaxis to the relaxation caused by endothelin 1.

Influence of the NOS inhibitor L-N^G-nitro-arginine (LNNA) AND THE SELECTIVE NEURONAL NITRIC OXIDE SYNTHASE (NNOS) INHIBITOR 1-(2-TRIFLUOROMETHYLPHENYL) IMIDAZOLE (TRIM) ON THE ACTIONS OF ENDOTHELIN 1. The NOS inhibitor L-NNA (3 × 10⁵ M) that caused maximal suppression of the NANC nerve-mediated IAS relaxation (Rattan and Chakder, 1992) had no significant effect on the contractile actions of endothelin 1, but it caused a significant attenuation of the IAS smooth muscle relaxation by endothelin 1 (Fig. 2). It is well known that L-NNA, being a general NOS inhibitor, may not be a good agent to discriminate the involvement of a specific type of NOS, i.e., neuronal or brain (nNOS), endothelial NOS, and inducible NOS in a given system (Moore and Handy, 1997). To specifically investigate the role of nNOS in the mediation of inhibitory effects of endothelin 1 in the IAS, we examined the influence of a relatively selective nNOS inhibitor TRIM (Handy and Moore, 1997; Moore and Handy, 1997) on the effects of endothelin 1. First, we examined the influence of TRIM on the NANC nerve-mediated relaxation of the IAS smooth muscle. Data given in Fig. 3 show that TRIM causes a significant and concentration-dependent attenuation of NANC nerve-mediated relaxation of the IAS. In control experiments, the fall in the IAS tension with 0.5 and 1 Hz EFS was 46.4 ± 5.4 and 62.5 ± 4.5%, respectively; that was significantly attenuated to 23.3 ± 4.7 and 41.5 ± 6.3%, respectively, following TRIM (3 × 10⁴ M) (p < .05; n = 8). The fall in the basal tension of the IAS caused by NANC nerve stimulation by the higher frequencies of EFS was likewise attenuated by TRIM.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Influence of the NOS inhibitor L-NNA on the biphasic effect of endothelin 1 on the basal IAS tone. Data (mean and S.E.) show a significant blockade of the inhibitory (*, p < .05; n = 11) but not the excitatory (p > .05; n = 11) effect of endothelin 1 on the basal tension of the IAS smooth muscle in the presence of the NOS inhibitor L-NNA.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Data (mean and S.E.) demonstrate the attenuation of the IAS smooth muscle relaxation by NANC nerve stimulation by different frequencies of EFS in the presence of different concentrations of the selective nNOS inhibitor TRIM (3 × 104 M). The attenuation of the IAS relaxation by TRIM was significant and concentration-dependent (*, p < .05; n = 8).

4

4

7

6

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Differential influence of TRIM on the excitatory versus the inhibitory effects of endothelin 1 on the IAS. Data (mean and S.E.) shows a significant blockade of the inhibitory (*, p < .05; n = 5) but not the excitatory effect (p > .05; n = 5) of endothelin 1 on the basal tension of the IAS smooth muscle in the presence of the nNOS inhibitor TRIM.

Influence of the N-Type Neuronal Ca⁺⁺ Channel Blocker -Conotoxin GVIA on the Biphasic Effect of Endothelin 1 on the IAS. First, we examined the effect of -conotoxin GVIA on the NANC nerve-mediated relaxation of the IAS smooth muscle. It was determined that the concentration of 1 × 10⁵ M was optimal to inhibit EFS-induced relaxation of the IAS. Under these experimental conditions, the toxin was found to cause a significant blockade of the IAS smooth muscle relaxation but not the contraction caused by endothelin 1 (Fig. 5; p > .05; n = 5). This concentration of the toxin was found to have no adverse effect on the basal tone of the IAS, 22.8 ± 2.9 in control experiments versus 24.2 ± 3.2 mN in the presence of the toxin (p > .05; n = 5).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effect of -conotoxin GVIA on the biphasic effect of endothelin 1 on the IAS. The toxin caused a significant blockade of the fall (*, p < .05; n = 5) but not of the rise (p > .05; n = 5) in the basal tension of the IAS.

Influence of the Protein Kinase C (PKC) Inhibitor 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and the Calmodulin Inhibitor N-(4-Aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13) on the IAS Contraction Caused by Endothelin 1. Because the activation of PKC (Bitar et al., 1991; Hillemeier et al., 1996; Sohn et al., 1997) and calmodulin (Hillemeier et al., 1991; Yu et al., 1995) have been shown to be the two major pathways involved in the basal tone and in the agonist-induced contraction of the sphincteric smooth muscle, we examined the influence of the commonly used PKC and calmodulin inhibitors H-7 and W-13, respectively, on the increase in the basal IAS tone by endothelin 1. H-7 and W-13 in the appropriate concentrations (3 × 10⁷ and 1 × 10⁶ M), when used alone caused a significant attenuation of the rise in the basal tone of the IAS by endothelin 1 (Fig. 6). To explore the involvement of PKC and calmodulin pathways in the mediation of contractile response in parallel, we examined the influence of these inhibitors in combination. Interestingly, the combination had no greater attenuation of the contractile response of endothelin 1 when compared to the individual inhibitors (p > .05; n = 5; Fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Influence of the calmodulin inhibitor W-13 (upper panel) and the protein kinase C inhibitor H-7 (middle panel) used alone, and in combination (lower panel), on the rise in the basal IAS tension by endothelin 1. The data (mean and SE) show a significant blockade of the control excitatory effect (*, p < .05; n = 5) of endothelin 1 on the basal tension of the IAS smooth muscle by either H-7 or W-13. The combination of H-7 and W-13 caused no further attenuation of endothelin 1-induced rise in IAS tension.

7

7

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Influence of W-13 (1 × 106 M) on the fall in the basal tension of the IAS caused by endothelin 1 (1 × 107 M). The data (mean and S.E.) show a significant suppression of the IAS smooth muscle relaxation in the presence of W-13 (*, p < .05).

Influence of Endothelin 1 Tachyphylaxis on the Actions of Endothelin 2 in the IAS. Endothelin 1 tachyphylaxis caused a significant diminution of both the relaxant and contractile actions of endothelin 2 on the IAS smooth muscle (p < .05; n = 5; Fig. 8). In control experiments, the initial fall in the basal tension of the IAS with 1 × 10⁷ and 1 × 10⁶ M endothelin 2 were 18.3 ± 6.3 and 57.3 ± 2.6%, respectively. The later rise in the IAS tension with the same concentrations of endothelin 2 were 42.9 ± 7.7 and 65.5 ± 7.1%, respectively. The fall in the basal tension of the IAS caused by 1 × 10⁷ and 1 × 10⁶ M endothelin 2 in the presence of endothelin 1 tachyphylaxis were 4.1 ± 2.5 and 3.7 ± 2.5%, respectively. The rise in the IAS tension caused by the same concentrations of endothelin 2 in the presence of endothelin 1 tachyphylaxis were 6.8 ± 4.3 and 2.0 ± 2.0%, respectively (p < .05; n = 5; Fig. 8).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Influence of endothelin 1 tachyphylaxis on the biphasic effect of endothelins 2 on the basal IAS tension. Note that endothelin 1 tachyphylaxis caused almost complete obliteration of the fall as well as the rise in the IAS tension caused by not only endothelin 1 but also by endothelin 2 (*, p < .05; n = 5).

	Discussion

Top Abstract Introduction Results Discussion References

The present studies on the basal tone of the IAS smooth muscle show a biphasic effect of endothelins on the spontaneously tonic smooth muscle of the gastrointestinal tract. The biphasic action of endothelin 1 consisted of an initial brief relaxation followed by a sustained contraction.

The initial relaxant action of endothelin 1 was found to be via the activation of nNOS at the myenteric nerve terminals of the IAS. Among all of the neurohumoral antagonists investigated, the relaxant action of endothelin 1 was only blocked by the NOS inhibitors, N-type neuronal Ca⁺⁺ channel blocker -conotoxin GVIA and the calmodulin inhibitor W-13. The neurotoxin TTX and cycloxygenase inhibitor indomethacin were found to have no effect on the endothelin response. We (Chakder and Rattan, 1996) and others (Mashimo et al., 1996) have shown previously that a part of the relaxant action of vasoactive intestinal polypeptide that was not affected by the neurotoxin TTX, occurs via the activation of specific receptors on the nerve terminals of the myenteric neurons. The data suggest that the relaxant action of endothelin occurred via the activation of endothelin receptor below the axonal level. The studies by Kitsukawa et al. (1994) on the isolated smooth muscle cells further corroborate the thesis that the relaxant action of endothelin 1 is indirect. To further verify the involvement of nNOS in the inhibitory action of endothelin, we examined the influence of a selective nNOS inhibitor TRIM. Interestingly, TRIM, like L-NNA, caused near obliteration of the IAS smooth muscle relaxation caused by endothelin 1.

It is well known that calmodulin plays an important role in the activation of constitutive nNOS (Matsuoka et al., 1994; Stevens-Truss et al., 1997). We found that the calmodulin inhibitor W-13, in the concentrations known to inhibit calmodulin, caused near obliteration of the fall in the basal tone caused by endothelin 1. These data further corroborate the involvement of nNOS in the mediation of the IAS smooth muscle relaxation by endothelin 1.

The exact intracellular pathway of the IAS smooth muscle relaxation by endothelin was not investigated in the present study. However, in general, it is well known that the smooth muscle relaxation involving nNOS occurs primarily via the activation of guanylate cyclase and an increase in the cytosolic cyclic GMP (Chakder and Rattan, 1993b; Luo et al., 1995; Franck et al., 1997).

The contractile action of endothelin 1 unlike its relaxant action on the IAS was caued by the direct activation of endothelin receptor at the smooth muscle. This conclusion was inferred because none of the neurohumoral blocking agents investigated, including L-NNA and TRIM, had any significant effect on the IAS smooth muscle contraction caused by endothelin 1. The direct contractile action of endothelin 1 in the IAS smooth muscle was similar to that of the rat colon (Moummi et al., 1992) and guinea pig gastric smooth muscles (Kitsukawa et al., 1994), and other regions of the gastrointestinal tract (Okabe et al., 1995; Bitar et al., 1992).

For in-depth examination of the intracellular mechanism of action of endothelin in producing IAS smooth muscle contraction, we investigated the influence of specific inhibitors of two major pathways responsible for the smooth muscle contraction in the basal state as well as in response to certain agonists. For this, the influences of the calmodulin and PKC inhibitors W-13 and H-7, respectively (Biancani et al., 1994), on the IAS smooth muscle contraction by endothelin 1 were investigated. The data revealed the involvement of both pathways for smooth muscle contraction by endothelin 1 because both W-13 and H-7 caused substantial suppression of the smooth muscle contraction. We raised the issues whether both pathways are involved in the endothelin 1-mediated IAS smooth muscle contraction in parallel and whether these pathways together may account fully for the smooth muscle contraction by endothelin 1. Interestingly, the combination of W-13 and H-7 caused no further attenuation of the IAS smooth muscle contraction observed by the individual use of these agents. The observations suggest that the calmodulin and PKC pathways, for the endothelin-mediated IAS smooth muscle contraction, may lie in series rather than parallel. It is possible that PKC-activated smooth muscle contraction is mediated via the Ca⁺⁺-calmodulin pathway (Singer, 1990; Miura et al., 1997). The studies further raise the possibility of intracellular mechanism other than Ca⁺⁺-calmodulin and PKC for the remaining smooth muscle contraction by endothelin 1 in the presence of the combination of W-13 and H-7. The participation of exact intracellular mechanisms independent of PKC and Ca⁺⁺-calmodulin pathways responsible for the endothelin-induced contraction of the smooth muscle is not known. In this regard, the relative roles of phospholipase D activation independent of PKC activation, Ca⁺⁺ influx, release of intracellular Ca⁺⁺, Na⁺/H⁺ exchange, increased sensitivity to Ca⁺⁺, adenylate and guanylate cyclase inhibition, in the mediation of smooth muscle contraction remain to be determined.

To date, in general, two types of endothelin receptors (ET_A and ET_B) have been recognized to explain the actions of endothelins in different systems (Kitsukawa et al., 1994; Masaki et al., 1994; Rubanyi and Polokoff, 1994; Allcock et al., 1995; Gray et al., 1995; Okabe et al., 1995). There is some data to suggest that different receptor subtypes may mediate the inhibitory and excitatory smooth muscle responses to endothelin. ET_B receptor activation may mediate the smooth muscle relaxation, whereas both ET_A and ET_B may be involved in the smooth muscle contraction by endothelins (Kobari et al., 1994; Irie et al., 1995; Miasiro et al., 1995; Lucas et al., 1996; Higashi et al., 1997). Furthermore, it has been shown in different systems that the activation of ET_B may be associated with the activation of constitutive NOS (endothelial NOS or nNOS) and release of NO (Kobari et al., 1994; Lucas et al., 1996; Higuchi and Satoh, 1997). The earlier studies have shown that the brain contains exclusively the ET_B receptors (Lysko et al., 1995) and further suggest the possibility of endothelins-induced IAS smooth muscle relaxation via the activation of ET_B receptors on nNOS-containing nerve terminals.

The characterization of the endothelin receptor subtype/s in the smooth muscle contraction or relaxation was not within the scope of the present project. However, cross-tachyphylaxis between the relaxant and contractile actions of endothelin 1 and endothelin 2 suggest the involvement of a common receptor subtype/s in the responses of the two endothelins. Furthermore, our preliminary data with the selective antagonists of ET_A and ET_B BQ-123 and IRL-1038, respectively, show that these antagonists were almost equally effective in blocking the responses to endothelins 1 and 2. The influence of these antagonists on the relaxant responses of the endothelins were not investigated. The relative involvement of ET_A and ET_B receptors in the endothelin-mediated relaxation and contractile responses remains to be determined.

Although, there are numerous studies in different smooth muscle systems, including the gastrointestinal tract, to report the contractile actions of endothelins, there is limited data that report the smooth muscle relaxation by endothelin. In general, the studies reporting the smooth muscles relaxation by endothelins were done following their contraction by another agonist or on the spontaneously phasic smooth muscles. To our knowledge, the relaxant response of endothelin on the spontaneously tonic smooth muscle has not been reported before.

In summary, these studies show that endothelin causes the relaxation and contraction of the spontaneously tonic sphincteric gastrointestinal smooth muscle. The relaxation by endothelins was observed as an initial and transient response that was followed by the sustained contraction. The data suggest that the IAS smooth muscle relaxation by endothelin is primarily NOS-mediated by the activation of receptor at the myenteric nerve terminals. The IAS smooth muscle contraction in response to endothelin, on the other hand, may be caused by the direct activation of the smooth muscle cells. The data suggest the involvement of both Ca⁺⁺-calmodulin and PKC pathways for the smooth muscle contraction by endothelin. These pathways appear to lie in series rather than in parallel. The studies suggest important neuromodulatory influences of endothelins on the basal tone of the IAS smooth muscle. Future studies should focus on the localization of endothelins in the neuronal and non-neuronal structures of the IAS. Additional studies on the role of endothelins in the inhibitory neurotransmission and morphogenesis of the gastrointestinal tract may provide important information on the pathophysiology of the gastrointestinal motility disorders.