Internal Anal Sphincter Smooth Muscle Research Articles

Mechanism of action of angiotensin II (Ang II) in the internal anal sphincter (IAS) smooth muscle

Mechanism of action of angiotensin II (Ang II) in the internal anal sphincter (IAS) smooth muscle

Mechanism of action of angiotensin II (Ang II) in the internal anal sphincter (IAS) smooth muscle

Mechanism of action of angiotensin II (Ang II) in the internal anal sphincter (IAS) smooth muscle

Inducible and neuronal nitric oxide synthase involvement in lipopolysaccharide-induced sphincteric dysfunction.

We examined the effect of endotoxin lipopolysaccharide (LPS) on the basal tone and on the effects of different stimuli and agonists and transcriptional and translational expression of nitric oxide (NO) synthase (NOS) isozymes in the lower esophageal sphincter (LES), pyloric sphincter (PS), and internal anal sphincter (IAS). NO release was also examined before and after LPS. LPS caused a dose-dependent fall in the basal tone and augmentation of the relaxation caused by nonadrenergic, noncholinergic (NANC) nerve stimulation in the LES and IAS. In the PS, LPS had no significant effect on the basal tone and caused an attenuation of the NANC relaxation and an augmentation of the contractile response of muscarinic agonist. Interestingly, the smooth muscle relaxation by atrial natriuretic factor was suppressed in the LES and IAS but not in the PS. These changes in the sphincteric function following LPS may be associated with increase in the inducible NOS (iNOS) expression since they were blocked by iNOS inhibitor L-canavanine. Augmentation of NANC relaxation in the LES and IAS smooth muscle by LPS may be due to the increased activity of neuronal NOS and NO production.

Heme oxygenase activity in the internal anal sphincter: Effects of nonadrenergic, noncholinergic nerve stimulation

Background & Aims: To date, the exact role of carbon monoxide (CO) in the nonadrenergic, noncholinergic (NANC) relaxation is not known. This is partly related to the lack of an appropriate method to measure heme oxygenase (HO) activity in the gastrointestinal tissues. Methods: HO activity of the opossum internal anal sphincter (IAS) smooth muscle was determined using a newly developed assay system that used radiolabeled hemin as a substrate. Enzyme activity of the IAS tissues was measured in the basal state, after electric field stimulation (EFS), ganglionic stimulant dimethyl diphenyl piperazinium iodide (DMPP), and neuropeptide vasoactive intestinal polypeptide (VIP). The presence and localization of HO was examined by Western blot analysis and immunocytochemistry. Results: NANC nerve stimulation of the IAS smooth muscle by EFS (0.25–5 Hz), DMPP, and VIP caused a significant increase in the HO activity of the IAS. The increase in HO activity by EFS was inhibited by the HO inhibitor Tin protoporphyrin (1 × 10 −4 mol/L). Both HO-1 and HO-2 were present in the IAS tissue extracts, and both enzymes were localized in the neurons of the myenteric plexus. The method for HO activity determination used in the present study was found to be reliable and reproducible. Conclusions: The data suggest that the HO pathway may have a role in neurally mediated relaxation of the IAS. The exact site of involvement and the source of HO activity, however, remains to be determined. GASTROENTEROLOGY 2000;118:477-486

Mechanism of action of cholera toxin on the opossum internal anal sphincter smooth muscle.

Cholera toxin (CTX), an activator of G(s) protein, is an important pharmacological tool in G protein research. The effect and the mechanism of action of CTX in the gastrointestinal smooth muscle, including the internal anal sphincter (IAS), are not known. The present investigation was carried out to examine the effects of CTX on the signal transduction associated with the adenylate cyclase (AC) pathway on the basal tone of the IAS smooth muscle. CTX caused a prompt and dose-dependent fall in the basal tone of the IAS that was not affected by the neurotoxins TTX and omega-conotoxin or the nitric oxide synthase inhibitor N(G)-nitro-L-arginine. The cyclooxygenase inhibitor indomethacin, cAMP-dependent protein kinase inhibitor Rp-8-bromoadenosine 3',5' cyclic monophosphorothioate inhibited CTX-induced IAS smooth muscle relaxation. Furthermore, CTX caused a concentration-dependent relaxation of the isolated smooth muscle cells (SMC) of the IAS, which was blocked by G(s)alpha antibody (G(s)alpha-Ab). The IAS smooth muscle relaxation was accompanied with an increase in the GTPase activity that was also specifically blocked by G(s)alpha-Ab. We conclude that a major part of the inhibitory action of CTX in the IAS is via the direct response of the SMC that is linked with G(s) protein to the AC pathway. A part of the inhibitory action of CTX on the smooth muscle occurs via the activation of cyclooxygenase pathway. The relative contribution of such actions of CTX in the smooth muscle in the gastrointestinal motility disturbances following cholera infection remains to be determined.

Sites of actions of contractile and relaxant effects of pituitary adenylate cyclase activating peptide (PACAP) in the internal anal sphincter smooth muscle.

In summary, PACAP exerts a biphasic effect (an initial contraction followed by a relaxation) in the IAS. The initial contractile effect with higher concentrations of PACAP was found to be mediated by the activation of PACAP receptor at the substance P-containing nerve terminals. The PACAP receptor(s) responsible for the inhibitory action of the neuropeptide is(are) hypothesized to be present in the IAS smooth muscle cells and on the myenteric nerve terminals. The exact nature and the role of PACAP and the PACAP receptors in the inhibitory neurotransmission, the relationship of PACAP receptors with substance P-containing neurons and IAS smooth muscle cells, and interactions with the NOS pathway and VIP remain to be determined.

Involvement of pituitary adenylate cyclase-activating peptide (PACAP) in the internal anal sphincter (IAS) smooth muscle relaxation

Involvement of pituitary adenylate cyclase-activating peptide (PACAP) in the internal anal sphincter (IAS) smooth muscle relaxation

Brain-gut-liver-immune axis during non-inflammatory stress in rat

Patients with systemic sclerosis (SSc) have impairments in gastrointestinal smooth muscle function. The disorder has been associated with circulating antibodies to cholinergic muscarinic the type-3 receptor (M3-R). We investigated whether it is possible to neutralize these antibodies with pooled human IgGs (pooledhIgG).We studied the effects of IgGs purified from patients with SSc (SScIgGs) on cholinergic nerve stimulation in rat colon tissues. We also examined the effects of SScIgGs on M3-R activation by bethanechol (BeCh), M3-R occupancy, and receptor binding using immunofluorescence, immunoblot, and enzyme-linked immunosorbent analyses of human internal anal sphincter (IAS) smooth muscle cells, before and after administration of pooledhIgG. Functional displacement of M3-R occupancy by the SScIgGs was compared with that of other IgGs during the sustained phase of BeCh-induced contraction of intact smooth muscles from rats.SScIgG significantly attenuated neurally mediated contraction and acetylcholine release in rat colon as well as BeCh-induced sustained contraction of the IAS smooth muscle. In immunofluorescence analysis, SScIgG co-localized with M3-R. In immunoblot and enzyme-linked immunosorbent analyses, M3-R loop-2 peptide and human IAS SMC membrane lysates bound significant amounts of SScIgG, compared with IgGs from healthy individuals and pooledhIgG. Binding was attenuated significantly by application of pooledhIgG, which by itself had no significant effect. Incubation of samples with pooledhIgG, or mixing pooledhIgG with SScIgG before administration to tissues, significantly reduced binding of SScIgG, indicating that pooledhIgG prevents SScIgG blockade of M3-R.In studies of rat and human tissues, pooled human IgG prevent and reverses the cholinergic dysfunction associated with the progressive gastrointestinal manifestations of SSc by neutralizing functional M3-R antibodies present in the circulation of patients with SSc.

Neuronal NOS gene expression in gastrointestinal myenteric neurons and smooth muscle cells.

Nitric oxide synthase (NOS) has been characterized in different tissues, and its localization has been suggested in different neuronal tissues, including the myenteric neurons and other nonneuronal cells. The present study examined the distribution of the neuronal NOS (nNOS) mRNA in different tissues of the opossum gastrointestinal tract, internal anal sphincter (IAS) smooth muscle cells, and myenteric neurons using slot-blot and Northern blot hybridization techniques with a specific rat brain nNOS cDNA probe. Significant levels of nNOS gene expression were found in both smooth muscle cells and myenteric neurons of the opossum IAS. This finding was confirmed by reverse transcriptase-polymerase chain reaction analysis of the RNA obtained from cultured opossum IAS smooth muscle cells and myenteric neurons and also from human intestinal smooth muscle and neuroblastoma cell lines. Pyloric sphincter had the highest level of nNOS gene expression compared with other gastrointestinal tissues. There was no significant difference in the nNOS gene expression between other sphincteric and nonsphincteric tissues examined. The present study shows the presence of nNOS gene expression in both neurons and smooth muscle cells. The higher levels of nNOS gene expression in the pyloric sphincter compared with other tissues may have pathophysiological significance in some disease conditions.

L-citrulline recycling in opossum internal anal sphincter relaxation by nonadrenergic, noncholinergic nerve stimulation

BACKGROUND & AIMS: L-citrulline formed stoichiometrically along with nitric oxide (1:1) from L-arginine may be enzymatically converted to L- arginine. The possibility of L-citrulline recycling in the maintenance of nitrergic neurotransmission in the opossum internal anal sphincter (IAS) smooth muscle strips was investigated. METHODS: Responses to nonadrenergic, noncholinergic (NANC) nerve stimulation by electrical field stimulation (EFS) (either short-train or continuous stimulation) on the basal IAS tension were recorded before and after the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA), L-NNA plus L-citrulline, or L-arginine. During continuous EFS, when the basal IAS tone after the initial relaxation had recovered to almost pre-EFS levels, the effects of L-citrulline or L-arginine were examined before and after L- glutamine, which is a putative blocker of L-citrulline uptake. RESULTS: Inhibition of NANC nerve-mediated IAS relaxation by L-NNA was reversed by L-citrulline as well as L-arginine. L-Citrulline and L-arginine caused concentration-dependent relaxation of the IAS tone recovered during the prolonged EFS. L-Glutamine blocked the responses of L- citrulline but not of L-arginine. Furthermore, L-glutamine increased the speed of recovery of IAS tone during continuous EFS. CONCLUSIONS: L- citrulline recycling may be responsible for the maintenance of IAS relaxation during frequent short-train and prolonged NANC nerve stimulation. (Gastroenterology 1997 Apr;112(4):1250-9)

Primary degeneration of the internal anal sphincter as a cause of passive faecal incontinence

Faecal incontinence is usually attributed to pelvic-floor denervation of striated muscle or direct sphincter trauma. We have identified a cause of passive faecal incontinence related to degeneration of the internal anal sphincter smooth muscle, in the absence of denervation, structural damage, external-sphincter weakness, or sensory abnormalities. Patients were included on the basis of: passive faecal incontinence, no urge faecal incontinence, low anal pressure whilst at rest, normal anal-squeeze pressure, endosonographically confirmed circumferentially intact internal and external anal sphincters, and normal pudendal nerve terminal nerve latencies. In a second analysis done to assess the proportion of patients with this disorder, we recorded the cause of incontinence in consecutive patients seen during a 6-month period. 45 patients (35 women, median age 63 years, range 23-80 years) fulfilled the diagnostic criteria. Median duration of symptoms was 2 years (3 months to 20 years). Nine of the 35 women were nulliparous. The median resting anal pressure was 40 cm water (16-56 cm water, normal > 60 cm water). Endosonography revealed an internal sphincter that was thin and hyperechogenic, and had a poorly defined edge. The normal increase in the thickness of the internal anal sphincter with age was not seen. Anal-squeeze pressure, sensitivity, and pudendal nerve latencies were normal. In the second analysis the condition was identified in eight of 230 patients, representing 4% of new referrals. Primary degeneration of the internal anal sphincter smooth muscle is a discrete clinical condition causing passive faecal incontinence.

Inhibitory effect of CO on internal anal sphincter: heme oxygenase inhibitor inhibits NANC relaxation.

We examined the effect and role of CO in opossum internal anal sphincter (IAS) relaxation in response to nonadrenergic noncholinergic (NANC) nerve stimulation. Effects of NANC nerve stimulation on the IAS tension and second messengers (cAMP and cGMP) were examined before and after the selective heme oxygenase (HO) inhibitor zinc protoporphyrin IX (Zn PP-IX). The HO activity of the IAS smooth muscle was determined before and after NANC nerve stimulation. CO caused a concentration-dependent and tetrodotoxin-resistant fall in the resting tension of the IAS. The direct action of CO was confirmed by its relaxant action on the isolated smooth muscle cells. Furthermore, CO caused an increase in the tissue cGMP levels comparable to that observed with nerve stimulation. Zn PP-IX caused suppression of IAS relaxation caused by NANC nerve stimulation and vasoactive intestinal polypeptide (VIP) but not by peptide histidine-isoleucine and suppression of the increase in cGMP in response to NANC nerve stimulation. Zn PP-IX had no significant effect on the IAS responses to CO, nitric oxide (NO), and the beta-adrenoceptor agonist isoproterenol. The IAS responses to CO were not modified by the NO synthase inhibitor NG-nitro-L-arginine. Significant HO activity was detected in the IAS, which increased further in response to NANC nerve stimulation and VIP. The direct relaxant actions of CO and the suppression of NANC-mediated relaxation of the IAS by the HO inhibitor suggest the involvement of CO in the neurally mediated IAS relaxation.

Release of nitric oxide by activation of nonadrenergic noncholinergic neurons of internal anal sphincter

The purpose of the present study was to investigate the direct release of nitric oxide (NO) in response to the stimulation of nonadrenergic noncholinergic (NANC) nerves. The studies were performed on isolated smooth muscle strips of the opossum (Didelphis virginiana) internal anal sphincter (IAS). Electrical field stimulation (EFS) using the appropriate parameters caused a frequency-dependent fall in the resting tone of the IAS. The release of NO was measured directly by the chemiluminescence method. The stimulation of NANC neurons by EFS and the nicotinic stimulant 1,1-dimethyl-4-phenylpiperazinium (DMPP) caused IAS relaxation with an accompanying release of NO. The release of NO and the fall in the resting tension of IAS in response to lower frequencies of EFS and DMPP were abolished by pretreatment of the smooth muscles with the neurotoxin tetrodotoxin and the NO-synthase inhibitor NG-nitro-L-arginine (L-NNA). The obliteration of the release of NO and the IAS relaxation in the presence of L-NNA reversed to control levels by the addition of the NO precursor L-arginine. The effect of L-NNA and L-arginine on NO release and IAS relaxation was stereoselective, since D-NNA and D-arginine had no significant effect. Vasoactive intestinal polypeptide also caused release of NO from IAS smooth muscle strips, which was abolished by L-NNA. However, isoproterenol and atrial natriuretic factor caused IAS relaxation without any increase in NO release. In conclusion, these studies demonstrate the direct release of NO in response to the stimulation of NANC inhibitory neurons of the gut.

Role of nitric oxide as a mediator of internal anal sphincter relaxation

The studies were performed in in vitro to examine the role of nitric oxide (NO) in nonadrenergic noncholinergic (NANC) nerve-mediated relaxation of the internal anal sphincter (IAS) smooth muscle strips of opossums. NO caused a concentration-dependent fall in the resting tension of the IAS. The inhibitory action of NO may be exerted directly on the IAS smooth muscle since it was not modified by the neurotoxin tetrodotoxin (1 x 10(-6) M), which abolished the neurally mediated fall in the IAS tension. The inhibitor of NO synthesis NG-nitro-L-arginine (L-NNA) produced concentration-dependent suppression of the neurally mediated fall in the IAS tension. The suppression of the neurally mediated IAS relaxation was stereoselective because D-NNA had no effect on the control responses. The suppressant action of L-NNA was selectively reversed by L-arginine in a concentration-dependent manner. The reversal was complete with 3 x 10(-4) M L-arginine. D-Arginine on the other hand, at the same concentration had no effect on L-NNA-suppressed IAS relaxation. Interestingly, the fall in the IAS tension caused by vasoactive intestinal polypeptide (VIP) (an inhibitory neurotransmitter in the IAS) was also inhibited by L-NNA (3 x 10(-5) M). From these data we conclude that NO or NO-like substances serve as important inhibitory mediators for the NANC nerve-mediated IAS relaxation. A part of the inhibitory action of VIP on the IAS involves NO-synthase pathway. The exact site of formation and release of NO or NO-like substances in response to NANC nerve stimulation remain to be investigated.

CGRP and ANF cause relaxation of opossum internal anal sphincter via different mechanisms

This investigation examined and compared the role of cyclic nucleotides in the mediation of internal anal sphincter (IAS) relaxation caused by the addition of neuropeptide calcitonin gene-related peptide (CGRP) and atrial natriuretic factor (ANF). The studies were performed in vitro on smooth muscle strips of opossum IAS. The relaxation produced by CGRP and ANF was examined before and after the addition of tetrodotoxin (TTX) (1 x 10(-6)M). At this concentration, TTX did not have any significant effect on the relaxation produced by either CGRP or ANF, suggesting that these peptides act directly on the smooth muscle. Addition of CGRP (3 x 10(-6) M) produced the maximal relaxation and significantly increased cAMP content without changing cGMP. On the other hand, addition of ANF (3 x 10(-6) M) caused a similar fall in IAS tension that was accompanied by a significant elevation in cGMP without any change in cAMP content. The rises in the levels of cyclic nucleotides preceded the onset of fall in the resting tension of IAS. Our results demonstrate that CGRP and ANF relax isolated strips of opossum IAS by their action directly at the smooth muscle and that this relaxation is associated with an increase in cAMP and cGMP, respectively. The studies suggest the presence of both cAMP and cGMP pathways in the IAS and that the relaxation of IAS smooth muscle in response to different peptides may occur via a specific intracellular biochemical pathway.

Effects of galanin on the opossum internal anal sphincter: Structure-activity relationship

The present study was carried out to investigate the effects of porcine galanin-(1-29), N-terminal fragment galanin-(1-10), C-terminal fragment galanin-(15-29), and the middle fragment galanin-(7-16) on the spontaneous tension of the opossum internal anal sphincter and on the decrease in the resting internal anal sphincter tension in response to neural stimulation by electrical field stimulation. Galanin and galanin-(1-10) caused a concentration-dependent decrease in the resting tension of internal anal sphincter and an augmentation of the percent decrease in the resting tension with electrical field stimulation. Galanin-(15-29), on the other hand, produced an increase in the resting tension of the internal anal sphincter and had no effect on the electrical field stimulation-induced decrease in the resting tension. Galanin-(7-16) produced no significant effect on the internal anal sphincter. The decrease in the internal anal sphincter tension by galanin and galanin-(1-10) was partially antagonized by tetrodotoxin, whereas the increase in the internal anal sphincter tension caused by galanin-(15-29) was not modified by tetrodotoxin. In contrast to its effect in the internal anal sphincter, galanin caused an increase in the resting tension and suppressed a decrease in the lower esophageal sphincter tension in response to electrical field stimulation. From these findings we conclude that (a) galanin exerts an inhibitory effect on the internal anal sphincter by activating galanin receptors both at the intramural inhibitory neurons and at the internal anal sphincter smooth muscle and that the N-terminal portion of galanin may be responsible for these actions; (b) the contractile action of galanin is produced by its action on the smooth muscle; and (c) the actions of galanin on the gastrointestinal tract are tissue specific.

Role of neuropeptide Y in opossum internal anal sphincter

The present investigation was undertaken to examine the effects and role of neuropeptide Y (NPY) on the internal anal sphincter (IAS). The studies were performed in alpha-chloralose-anesthetized opossums. Resting pressure in the IAS (IASP) was recorded using low-compliant continuously perfused catheters. The effect of NPY administered close intra-arterially on the resting IASP was examined before and after different neurohumoral antagonists and the neurotoxin tetrodotoxin (TTX). To examine the influence of sympathetic nerves, the ventral roots of lumbar sympathetic nerves were isolated in some experiments and stimulated electrically. The influence of NPY on the IAS relaxation in response to rectoanal reflex mimicked by rectal balloon distension (RD) was also examined. NPY caused dose-dependent rise in the IASP, which was not modified by different neurohumoral antagonists and TTX. The rise in IASP by electrical stimulation of ventral lumbar nerve root (L3) was suppressed but not abolished by phentolamine. However, guanethidine (which depletes both norepinephrine and NPY) caused complete obliteration of this response. The other effect of NPY on the IAS was significant antagonism of IAS relaxation caused by RD. From these results, we conclude that NPY exerts two different actions on the IAS. First, it causes a rise in IASP by its action directly at the IAS smooth muscle. Sympathetic nerve stimulation may cause a rise in IASP by its action directly at the IAS smooth muscle partially through release of NPY. Second, it inhibits IAS relaxation produced by rectoanal reflex. These findings would suggest an important regulatory role of NPY in rectoanal physiology.

Peptide Histidine Isoleucine and Vasoactive Intestinal Polypeptide Cause Relaxation of Opossum Internal Anal Sphincter via Two Distinct Receptors

The purpose of this investigation was to characterize the nature of peptide histidine isoleucine (PHI) and vasoactive intestinal polypeptide (VIP) receptors, and to examine the role of PHI in internal anal sphincter (IAS) relaxation. The studies were performed on opossums anesthetized with alpha-chloralose. The pressures in the IAS were recorded using continuously perfused catheters. The IAS responses to PHI analogues, PHI-27, PHM-27, PHI-(14-27)-NH2, PHI-(1-13), to VIP, to rectal balloon distention, sacral nerve stimulation, and local intramural stimulation were evaluated before and after PHI-(14-27)-NH2, PHI tachyphylaxis, and the VIP antagonists [4 Cl-D-Phe6, Leu17] VIP (VIP analogue) and (N-Ac-Tyr1, D-Phe2)-GRF(1-29)-NH2 (growth hormone releasing factor analogue). The inhibitory responses by all of the PHI analogues and VIP were not modified by tetrodotoxin. PHI-(14-27)-NH2 and PHI tachyphylaxis caused significant antagonism of the fall in internal anal sphincter pressure by PHI-27 and PHM-27 without modifying the IAS responses to VIP and rectal balloon distention, sacral nerve stimulation, and local intramural stimulation. On the other hand, VIP and growth hormone releasing factor analogues caused significant antagonism of VIP responses without modifying the responses to PHI-27. We conclude that distinct PHI and VIP receptors are present in the IAS smooth muscle and that PHI may not play a significant role in the IAS relaxation via the rectoanal reflex.

Effect of methylene blue and N-ethylmaleimide on internal anal sphincter relaxation.

The present studies were performed in vitro to define the participation of regulatory cyclic nucleotides in the relaxation of internal anal sphincter (IAS) smooth muscle in response to neural stimulation by electrical field stimulation (EFS) vs. exogenous vasoactive intestinal peptide (VIP). EFS and VIP both caused relaxation of the resting tone in the opossum-isolated IAS smooth muscle strips. The addition of permeant cyclic nucleotide derivatives, the guanylate cyclase stimulant sodium nitroprusside (SNP), and the adenylate cyclase stimulant forskolin caused a dose-dependent fall in the resting tension of IAS smooth muscle. The inhibitory effect of the agonists on the IAS smooth muscle was not modified by tetrodotoxin (TTX), a neurotoxin. TTX almost abolished the IAS responses to EFS. The effects of SNP and forskolin were selectively blocked by the putative inhibitors of corresponding enzyme systems, i.e., methylene blue (MB) (3 X 10(-5) M) for guanylate cyclase and N-ethylmaleimide (NEM) (10(-4) M) in the case of adenylate cyclase. NEM and not MB caused significant antagonism of the fall in IAS tension in response to both EFS and VIP during the control experiments. Such data suggest a common biochemical link (adenosine 3',5'-cyclic monophosphate as second messenger system) between the IAS smooth muscle relaxations with neural stimulation and VIP. In addition, a part of the IAS smooth muscle relaxation in response to EFS also involves the mediation of guanosine 5'-cyclic monophosphate.

Influence of purinoceptors' agonists and antagonists on opossum internal anal sphincter.

Studies were performed in alpha-chloralose-anesthetized and pancuronium-treated opossums. Resting internal anal sphincter pressures (IASP) were monitored using low-compliant continuously perfused catheters. P1 and P2 purinoceptor agonists, adenosine and ATP, respectively, administered close intra-arterially, caused dose-dependent decreases in the IASP. The inhibitory effect of these agonists on the IASP was tetrodotoxin resistant. Rectal balloon distension (RD) (which mimics the rectoanal inhibitory reflex) caused volume-dependent IAS relaxation. Electrical stimulation of the sacral nerve (SNS) also produced frequency-dependent decreases in IASP. The inhibitory response to adenosine (P1 purinoceptor agonist), ATP (P2 purinoceptor agonist), RD, and SNS on the internal anal sphincter (IAS) was examined before and after 8-phenyltheophylline (P1 purinoceptor antagonist) and alpha,beta-methylene ATP (P2 purinoceptor antagonist that irreversibly binds and desensitizes P2 purinoceptor). P1 and P2 purinoceptor antagonists produced selective antagonism of the inhibitory responses on the IAS of their respective agonists only. Furthermore, high doses of adenosine and ATP produced desensitization and block of their own actions only. The purinoceptors' antagonists, and the desensitization of purinoceptors by high doses of adenosine and ATP, failed to modify the fall in IASP in response to RD or SNS. From these studies we conclude that distinct inhibitory P1 and P2 purinoceptors are present on the IAS smooth muscle. However, these inhibitory purinoceptors may not be responsible for the rectoanal reflex-mediated IAS relaxation.