Direct and indirect innervation of smooth muscle cells of rat stomach,with special reference to the interstitial cells of Cajal

Interstitial cells of Cajal in the circular (ICC-CM) and longitudinal (ICC-LM) muscle layer of the rat gastric antrum and their innervation were studied ultrastructurally. Both ICC-CM and ICC-LM are characterized by many mitochondria, rough and smooth endoplasmic reticulum, caveolae, and formation of gap junctions with each other and with muscle cells, though ICC-LM tend to show more variable cytoplasmic features depending on section profiles. Close contacts between nerve terminals and both ICC-CM and ICC-LM are observed. These possible synaptic structures are characterized by: (1) accumulation of synaptic vesicles in nerve varicosities, (2) a narrow gap (about 20 nm) between pre- and postjunctional membranes, (3) lack of a basal lamina between pre- and postjunctional membranes, and (4) the presence of an electron-dense lining on the inner aspect of prejunctional membranes. Almost the same characteristics are observed between the nerve terminals and the muscle cells of both circular and longitudinal muscle layers of the same specimens. Therefore, we conclude that the smooth muscle cells of both circular and longitudinal layers of the rat antrum are directly and indirectly innervated via ICC. Their functional significance is discussed.     

Activation of intestinal smooth muscle cells by interstitial cells of Cajal in simulation studies

Activation of a two-dimensional sheet network (5 parallel chains of 5 cells each) of simulated intestinal smooth muscle cells (SMCs) by one interstitial cell of Cajal (ICC) was modeled by PSpice simulation. The network of 25 cells was not interconnected by gap-junction channels; instead, excitation was transmitted by the electric field that develops in the junctional clefts (JC) when the prejunctional membrane fires an action potential (AP). Transverse propagation between the parallel chains occurs similarly. The ICC cell was connected to cell E5 of the network [5th cell of the 5th (E) chain] via a high-resistance junction. The stimulating current, applied to the ICC cell interior, was made to resemble the endogenous undershooting slow wave (I(SW)). An I(SW) of 2.4 nA (over a rise time of 4 ms) took the ICC cell from a resting potential (RP) of -80 mV to a membrane potential of -41 mV. The slow wave produced a large negative cleft potential in the JC (V(JC); ICC-E5). The V(jc) brought the postjunctional membrane of E5 to threshold, causing this cell to fire an AP. This, in turn, propagated throughout the SMC network. If the ICC cell was given an RP of -55 mV (like SMC) and a slow wave of 40 mV amplitude (I(SW) of 1.8 nA), it still activated the SMC network. This was also true when the ICC cell was made excitable (developing an overshooting, fast-rising AP). In summary, one ICC cell displaying a slow wave was capable of activating a network of SMC in the absence of gap junctions.     

Proteins of interstitial cells of Cajal and intestinal smooth muscle, colocalized with caveolin-1

The murine jejunum and lower esophageal sphincter (LES) were examined to determine the locations of various signaling molecules and their colocalization with caveolin-1 and one another. Caveolin-1 was present in punctate sites of the plasma membranes (PM) of all smooth muscles and diffusely in all classes of interstitial cells of Cajal (ICC; identified by c-kit immunoreactivity), ICC-myenteric plexus (MP), ICC-deep muscular plexus (DMP), ICC-serosa (ICC-S), and ICC-intramuscularis (IM). In general, all ICC also contained the L-type Ca(2+) (L-Ca(2+)) channel, the PM Ca(2+) pump, and the Na(+)/Ca(2+) exchanger-1 localized with caveolin-1. ICC in various sites also contained Ca(2+)-sequestering molecules such as calreticulin and calsequestrin. Calreticulin was present also in smooth muscle, frequently in the cytosol, whereas calsequestrin was present in skeletal muscle of the esophagus. Gap junction proteins connexin-43 and -40 were present in circular muscle of jejunum but not in longitudinal muscle or in LES. In some cases, these proteins were associated with ICC-DMP. The large-conductance Ca(2+)-activated K(+) channel was present in smooth muscle and skeletal muscle of esophagus and some ICC but was not colocalized with caveolin-1. These findings suggest that all ICC have several Ca(2+)-handling and -sequestering molecules, although the functions of only the L-Ca(2+) channel are currently known. They also suggest that gap junction proteins are located at sites where ultrastructural gap junctions are know to exist in circular muscle of intestine but not in other smooth muscles. These findings also point to the need to evaluate the function of Ca(2+) sequestration in ICC.     

Development of the enteric nervous system,smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract

The generation of functional neuromuscular activity within the pre-natal gastrointestinal tract requires the coordinated development of enteric neurons and glial cells, concentric layers of smooth muscle and interstitial cells of Cajal (ICC). We investigated the genesis of these different cell types in human embryonic and fetal gut material ranging from weeks 4–14. Neural crest cells (NCC), labelled with antibodies against the neurotrophin receptor p75^NTR, entered the foregut at week 4, and migrated rostrocaudally to reach the terminal hindgut by week 7. Initially, these cells were loosely distributed throughout the gut mesenchyme but later coalesced to form ganglia along a rostrocaudal gradient of maturation; the myenteric plexus developed primarily in the foregut, then in the midgut, and finally in the hindgut. The submucosal plexus formed approximately 2–3 weeks after the myenteric plexus, arising from cells that migrated centripetally through the circular muscle layer from the myenteric region. Smooth muscle differentiation, as evidenced by the expression of -smooth muscle actin, followed NCC colonization of the gut within a few weeks. Gut smooth muscle also matured in a rostrocaudal direction, with a large band of -smooth muscle actin being present in the oesophagus at week 8 and in the hindgut by week 11. Circular muscle developed prior to longitudinal muscle in the intestine and colon. ICC emerged from the developing gut mesenchyme at week 9 to surround and closely appose the myenteric ganglia by week 11. By week 14, the intestine was invested with neural cells, longitudinal, circular and muscularis mucosae muscle layers, and an ICC network, giving the fetal gut a mature appearance.A.S.W. is funded by a PhD studentship awarded to A.J.B. by the Child Health Research Appeal Trust.     

Phenotypic changes in the regenerating rabbit bladder muscle. Role of interstitial cells and innervation on smooth muscle cell differentiation

 We have studied the phenotypic changes in regenerating smooth muscle (SM) tissue of detrusor muscle after local application of a necrotizing, freeze–thaw injury to the serosal surface of rabbit bladder. Bromo-deoxyuridine (BrdU) incorporation and immunofluorescence studies were performed on bladder cryosections from day 2 up to day 15 after surgery with monoclonal antibodies specific for some cytoskeletal markers [desmin, vimentin, non-muscle (NM) myosin] and for SM-specific proteins (α-actin, myosin, and SM22). Four days after lesion, some clls incorporated in regenerating SM bundles are BrdU positive, but all display a phenotypic pattern identical to that of the interstitial, highly proliferating cells, i.e., expression of vimentin. By days 7–15 the differentiation profile of regenerating SM returns to that of uninjured SM tissue (appearance of desmin, SM-type α-actin, and SM myosin). A chemical denervation achieved by 6-hydroxydopamine treatment for 2 weeks induces the formation of vimentin/SM α-actin/NM myosin/SM22-containing myofibroblasts in the interstitial, fibroblast-like cells of uninjured bladder. In the bladder wall, alteration of reinnervation during the regenerating SM process produces: (1) in the outer region, the activation of vimentin/SM α-actin/desmin myofibroblasts in the de novo SM cell bundles; and (2) in the inner region of bladder, including the muscularis mucosae, the formation of proliferating, fully differentiated SM cells peripherally to newly formed SM cell bundles. These findings suggest that: (1) the de novo SM tissue formation in the bladder can occur via incorporation of interstitial cells into growing SM bundles; and (2) the alteration of reinnervation during the regenerating process induces a spatial-specific differentiation of interstitial myofibroblasts in SM cells before SM cell bundling. Accepted: 14 May 1997     

Inflammatory gene expression by human colonic smooth muscle cells

Intestinal mucosal cells and invading leukocytes produce inappropriate levels of cytokines and chemokines in human colitis. However, smooth muscle cells of the airway and vasculature also synthesize cytokines and chemokines. To determine whether human colonic myocytes can synthesize proinflammatory mediators, strips of circular smooth muscle and smooth muscle cells were isolated from human colon. Myocytes and muscle strips were stimulated with 10 ng/ml of IL-1beta, TNF-alpha, and IFN-gamma, respectively. Expression of mRNA for IL-1beta, IL-6, IL-8, and cyclooxygenase-2 (COX-2) was induced within 2 h and continued to increase for 8-12 h. Regulated on activation, normal T cell-expressed and -secreted (RANTES) mRNA expression was slower, appearing at 8 h and increasing linearly through 20 h. Expression of all five mRNAs was inhibited by 0.1 microM MG-132, a proteosome inhibitor that blocks NF-kappaB activation. Expression of IL-1beta, IL-6, IL-8, and COX-2 mRNA was reduced by 30 microM PP1, an Src family tyrosine kinase inhibitor, and by 25 microM SB-203580, a p38 MAPK inhibitor. MAPK/extracellular regulated kinase-1 inhibitor PD-98059 (25 microM) was much less effective. In conclusion, human colonic smooth muscle cells can synthesize and secrete interleukins (IL-1beta and IL-6) and chemokines (IL-8 and RANTES) and upregulate expression of COX-2. Regulation of cytokine, chemokine, and COX-2 mRNA depends on multiple signaling pathways, including Src-family kinases, extracellular regulated kinase, p38 MAPKs, and NF-kappaB. SB-203580 was a consistent, efficacious inhibitor of inflammatory gene expression, suggesting an important role of p38 MAPK in synthetic functions of human colonic smooth muscle.     

Cytodifferentiation of the interstitial cells of Cajal of mouse colonic circular muscle layer. An EM study from fetal to adult life

Cytodifferentiation of the interstitial cells of Cajal related to the plexus entericus extremus and located on the inner face of the circular muscle layer of the mouse colon was studied in fetuses at term, unfed neonates, suckling and weaning young animals. In fetuses at term, the interstitial cells of Cajal are not found and their precursor cells are not identifiable among the cells present in the submucosal area facing the circular muscle layer, i.e., undifferentiated cells (mainly close to nerve fibers) and fibroblast-like cells (contacting each other and the developing smooth-muscle cells). Cells putatively considered as precursors of interstitial cells of Cajal, fibroblast-like cells rich in mitochondria, are present in unfed neonates (in small numbers) and in suckling animals (in large numbers). Differentiating interstitial cells of Cajal are undoubtedly recognizable during the second week of postnatal life (suckling period). Some of them have mixed fibroblastic- and interstitial-cell features and some have many interstitial-cell features. Both these cell types are already in contact with each other and differentiating and differentiated smooth-muscle cells. The undifferentiated cells, still present in unfed neonates, envelop the nerve fibers and differentiate as Schwann cells during the suckling period. During this period, as the nerve endings of the plexus entericus extremus develop, they immediately contact the differentiating interstitial cells of Cajal. During the weaning period, these cells are quite well differentiated although they do not have the same morphology as in adult animals before 30 days of age. Concomitantly, the nerve endings of the plexus entericus extremus contain an increasing number of synaptic vesicles.     

Smooth muscle cells, interstitial cells of Cajal and myenteric plexus interrelationships in the human colon

The plane between longitudinal and circular muscle of human colon, as revealed on examination with light and electron microscopes, has no clear-cut border. Some groups of smooth muscle cells, obliquely oriented and with features similar to both circular and longitudinal ones--the connecting muscle bundles--run from one muscle layer to another. Other groups of smooth muscle cells, possessing their own specific ultrastructural features--the myenteric muscle sheaths--, make up envelopes of variable thickness around some myenteric ganglia and nerve strands, partially or completely embedding them in one or other muscle layer. Non-neuronal, non-muscular cells (interstitial cells of Cajal, covering cells, fibroblast-like and macrophage-like cells) complicate the texture of the myenteric muscle sheaths, creating an intricate, interconnected cellular network inside them, widespread among nerve bundles and smooth muscle cells; however, only interstitial cells have cell-to-cell junctions also with the smooth muscle cells and nerve endings. These data document the existence in this colonic area of two different types of muscle cell arrangements, one of which, the myenteric muscle sheath, only contains putative pacemaker cells.     

Immunomagnetic enrichment of interstitial cells of Cajal

Disruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was approximately 63-fold and approximately 8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca(2+) or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells.     

Comparative study of interstitial cells of Cajal

The cells present in the alimentary canal, contacting both nerve endings and smooth muscle cells and named interstitial cells of Cajal, show different ultrastructural features. A comparative study has been performed in order to see if these differences can be related to the animal species studied or to the interstitial cell localizations inside the muscle wall of the various levels of the alimentary canal or to their contacts with other cells. Only mammals were considered, and rat, mouse, hedgehog and man have been studied. All the localizations where interstitial cells of Cajal have usually been found were considered: esophagus (body and lower sphincter), stomach (gastric extent of the lower esophageal sphincter, fundus and corpus), small intestine and colon. From this comparison a correlation was found between the morphology and the location of interstitial cells. On the contrary, the morphological differences existing between animal species do not seem to be that consistent. Moreover, the number of contacts between interstitial cells and between these and smooth muscle cells and nerve endings varies according to the interstitial cell location and morphology. It is concluded that the chain nerve endings----interstitial cells of Cajal----smooth muscle cells is not morphologically identical at each gastrointestinal level, and this finding is considered very important in interpreting the role played by the interstitial cells of Cajal in gastrointestinal motility.     

Volume-activated chloride currents in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) undergo marked morphological changes on contraction of the musculature, making it essential to understand properties of mechanosensitive ion channels. The whole cell patch-clamp technique was used to identify and to characterize volume-activated Cl- currents in ICC cultured through the explant technique. Hypotonic solutions (approximately 210 mosM) activated an outwardly rectifying current, which reversed near the equilibrium potential for Cl-. Time-dependent inactivation occurred only at pulse potentials of +80 mV, with a time constant of 478 +/- 182 ms. The degree of outward rectification was calculated using a rectification index, the ratio between the slope conductances of +65 and -55 mV, which was 13.9 +/- 1.5 at 76 mM initial extracellular Cl- concentration. The sequence of relative anion permeability of the outwardly rectifying Cl- channel was I- > Cl- > aspartate-. The chloride channel blockers, DIDS and 5-nitro-2-(3-phenlypropl-amino)benzoic acid, caused a voltage-dependent block of the outwardly rectifying Cl- current, inhibition occurring primarily at depolarized potentials. On exposure to hypotonic solution, the slope conductance significantly increased at the resting membrane potential (-70 mV) from 1.2 +/- 0.2 to 2.0 +/- 0.4 nS and at the slow-wave plateau potential (-35 mV) from 2.1 +/- 0.3 to 5.0 +/- 1.0 nS. The current was constitutively active in ICC and contributed to the resting membrane potential and excitability at the slow-wave plateau. In conclusion, swelling or volume change will depolarize ICC through activation of outwardly rectifying chloride channels, thereby increasing cell excitability.     

Protein kinases expressed by interstitial cells of Cajal

Interstitial cells of Cajal (ICC) are involved in the generation of electrical rhythmicity of intestinal muscle and in the transduction of neural inputs in the gut. Although the expression of receptors for neurotransmitters and hormones and some second messengers have been investigated in ICC, the protein kinases present in these cells have not been well documented. This study has demonstrated the immunohistochemical localisation of PKA, PKC and PKC in ICC that were identified by the known ICC marker, c-Kit, in the guinea-pig gut. Other PKCs, PKC , , , , , and , and Ca²⁺-calmodulin-dependent protein kinase II were not localised in ICC. Double labelling studies were conducted on longitudinal muscle–myenteric plexus and external muscle–myenteric plexus preparations of the oesophagus, stomach (fundus, corpus and antrum), duodenum, distal ileum, caecum, proximal and distal colon, and rectum. The three protein kinases were detected in c-Kit-immunoreactive ICC at the level of the myenteric plexus (IC-MY), in the muscle (IC-IM) and at the level of the deep muscular plexus (IC-DMP) in the small intestine. PKA was found in over 90% of IC-IM in all regions examined, and in over 90% of IC-MY in the gastric body and antrum and throughout the small and large intestines. PKC was in the majority of ICC in the gastric body and antrum and in the small intestine, but was largely absent from ICC in the oesophagus, proximal stomach and large intestine. PKC occurred in the majority of ICC in all regions except the rectum. The intensity of staining was greatest for PKA, with PKC giving comparatively weak labelling of ICC. PKA was also detected in myenteric neurons, smooth muscle, macrophages and fibroblast-like cells. PKC labelling occurred in large, multipolar neurons throughout the small and large intestine, as well as in lymph vessels and in capillaries. It is concluded that PKA, PKC and PKC are all present in ICC, with the differences in their localisations suggesting specific roles for each in ICC function.     

Structural characterization of interstitial cells of Cajal in myenteric plexus and muscle layers of canine colon

We have carried out a detailed ultrastructural study of the interstitial cells near the myenteric plexus of the canine colon and defined the structural characteristics which distinguish them from other resident non-neural cells. We have also examined the interconnections of these interstitial cells with nerves, the longitudinal muscle, and the circular muscle. In addition, we sought connections between interstitial cells of the myenteric plexus and those described earlier at the inner border of the circular muscle in proximal and distal colon. The interstitial cells of the myenteric plexus were structurally distinctive, and made gap junctions with one another and occasionally with smooth muscle. There seemed to be two subsets of these interstitial cells, one associated with the longitudinal muscle and the other with the circular muscle. Cells of both subsets were often close (less than or equal to 20 nm) to nerve profiles. The interstitial cells near the longitudinal muscle layer penetrated slightly into the muscle layer, but those near the circular muscle did not and neither set contacted the other. Moreover, interstitial cells of Cajal located near the myenteric plexus were never observed to contact those at the inner border of circular muscle. The interstitial cells of Cajal at the canine colon myenteric plexus are structurally organized to provide independent pacemaking activities for the longitudinal and adjacent circular muscle. Their dense innervation suggests that they mediate neural modulation of intestinal pacemaker activities. Moreover, they lack direct contacts with the interstitial cell network at the inner border of circular muscle, which is essential for the primary pacemaking activity of circular muscle. The structural organization of interstitial cells in canine colon is consistent with their proposed role in pacemaking activity of the two muscle layers.     

大鼠结肠平滑肌细胞的钙库操纵性通道

目的:探讨大鼠结肠平滑肌细胞是否存在钙库操纵性通道(SOC)。方法:荧光探针Fura-2/AM标记细胞内游离Ca2+后,用荧光分光光度计检测毒胡萝卜素(thapsigargin)和咖啡因(caffeine)耗竭胞内钙库后激活的SOC通道对酶解分离的大鼠结肠平滑肌细胞[Ca2+]i的影响。结果:在无Ca2+缓冲液中,thapsigargin(1μmol/L)以及caf-feine(10 mmol/L)分别使[Ca2+]i由静息时(68.32±3.43)nmol/L升高至(240.85±12.65)nmol/L(、481.25±34.77)nmol/L,继之,向细胞外液中引入两种浓度的Ca2+(1.5 mmol/L和3.0 mmol/L),导致[Ca2+]i进一步升高,分别为(457.55±19.80)nmol/L、(1005.93±54.62)nmol/L;(643.88±34.65)nmol/L、(920.16±43.25)nmol/L。且上述升高效应对维拉帕米(verapamil,5μmol/L)以及KCl引起的细胞膜去极化不敏感,但可被La3+(1 mmol/L)抑制。结论:在酶解分离的大鼠结肠平滑肌细胞上,存在胞内钙库耗竭激活的SOC通道,为支持在电兴奋性细胞上存在库容性Ca2+内流提供了实验和理论依据。     

Excitability of canine colon circular muscle disconnected from the network of interstitial cells of Cajal.

The 6 cpm omnipresent slow waves recorded in the circular muscle (CM) layer of canine colon are generated at the submucosal surface of the CM layer. After removal of the submucosal network of interstitial cells of Cajal (ICC), 66% of the CM preparations (25 of 38) were quiescent in Krebs solution. In the presence of carbachol, seven of nine of these spontaneously quiescent CM preparations demonstrated slow wave-like activity with mean frequency, duration and amplitude of 5.9 +/- 0.4 cpm, 2.8 +/- 0.5 s, and 0.8 +/- 0.2 mV, respectively. Similar slow wave-like activities were induced by TEA (seven out of eight quiescent CM preparations) with frequency, duration and amplitude of 6.1 +/- 0.2 cpm, 2.7 +/- 0.5 s, and 1.0 +/- 0.2 mV, respectively, and by BaCl2 (eight of eight quiescent CM preparations) with frequency, duration, and amplitude of 6.3 +/- 0.3 cpm, 1.8 +/- 0.2 s, and 0.5 +/- 0.1 mV, respectively. All the induced activities were abolished in the presence of 1 microM D600. CM preparations with the submucosal ICC network intact (ICC-CM) showed slow wave activity in Krebs solution at a frequency of 6.2 +/- 0.2 cpm, a duration of 3.6 +/- 0.2 s, and an amplitude of 1.0 +/- 0.1 mV (n = 22). When ICC-CM preparations were stimulated by BaCl2, carbachol, or TEA, the slow wave frequency did not change significantly, but the duration increased as well as the amplitude. In the presence of D600, the upstroke of slow waves remained and the frequency was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of inhibitory innervation in porcine colonic circular muscle

Effects of stimulation of intramural nerves in the circular smooth muscle layer of the porcine colon (Sus scrofa domestica) were studied using the sucrose-gap technique. Electrical field stimulation of the preparation, superfused with Krebs solution at 21 degrees C, induced a transient hyperpolarization of the smooth muscle cell membrane. This hyperpolarization was an inhibitory junction potential (IJP). The responses obtained from circular muscle originating from either the centripetal or centrifugal gyri of the ascending colon did not differ significantly. The IJP was characterized as being mediated by intramural, nonadrenergic, noncholinergic (NANC) nerves. The amplitude and latency of the IJP changed linearly with temperature (15-25 degrees C: +1 mV and -0.1 s per degree Celsius, respectively) reflecting a temperature-dependent synchronization of transmitter release. The membrane resistance decreased during the IJP. The IJP amplitude decreased or increased during conditioning hyperpolarizations or depolarizations, respectively, and reversed at membrane potentials about 30 mV more negative than the resting membrane potential. Potassium conductance blocking agents, barium (1 mM), tetraethylammonium chloride (TEA, 20 mM), 4-aminopyridine (4-AP, 5 mM), apamin (1 microM), and aminacrine (10(-4) M) added to the superfusion medium increased the membrane resistance. Only barium, TEA, and apamin depolarized the smooth muscle cell membrane. The IJP amplitude decreased in the presence of aminacrine and apamin to 75 and 35%, respectively, suggesting that apamin-sensitive Ca2+-activated K+ channels are involved in this response. ATP, adenosine, and related adenine nucleotides in concentrations up to 10(-3) M did not mimic the IJP. Superfusion with ATP for 15 min revealed a gradually increasing attenuation by up to 20% of the IJP. This might suggest that the release of neurotransmitter from intramural NANC nerves is modulated presynaptically via purinoceptors. Exogenously applied vasoactive intestinal polypeptide (VIP) in concentrations of 10(-9) to 10(-4) M did not affect the preparation. Also at elevated temperatures (up to 35 degrees C), VIP (10(-7) to 10(-4) M) did not cause measurable effects. It is concluded that the inhibitory mediator of the intramural NANC nerves present in the circular muscle layers of the porcine colon is neither a purine nor VIP.