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Nonadrenergic-noncholinergic (NANC) and cholinergic innervation in the circular muscle layer of human colon. An histochemical study
ABSTRACTS OF PAPERS
October 1994
NONADRENERGIC-NONCHOLINERGIC (NANC) AND CHOLINERGIC INNERVATION IN THE CIRCULAR MUSCLE STUDY. LAYER OF HUMAN COLON. AN HISTOCHEMICAL P.Matini and M.S.Faussone-Pellegrini. Dept.Hum.Anat. and Histol. University of Florence, Florence, Italy. Two differently arranged portions have been identified in the circular muscle layer of the human colon: a main (outer, CCL) and a submucosal(inner, ICL)portion.ICLhaspeculiarsmoothmusclecells and interstitial cells of Cajal arranged in a continuous layer, which is thicker at the cecocolonic junction (CCJ)than at right and left colon. Myogenic activity seems to origin at the circular muscle and the occurrence of spontaneous spasms have been observed at CCJ. Nerve fibers are present in the two circular muscle layers of the entire colon. The distribution of both NANC and cholinergic nerves either at ICL OT OCL of the entire human colon has been histochemically detected in order to see if these layers possess innervational differences related to their specific roles. Antibodies against Chat (Chemicon), VIP (Amersham), Nitric Oxide synthase (NOs, gift of dr. B.Mayer, University of Graz), NPY and Galanin (Affiniti) have been used; nerve distribution has been revealed by neuronal enolase immunoreactivity (Affiniti). Specimens of CCJ, right and left colon were surgically obtained (Prof.C.Cortesini, Surgery Clinic, University of Florence) from patients operated on for non stenosing colon carcinoma Duke’s A type. Identical distribution and richness in nerve fibers were observed at all areas examined, while differences in neurotransmitters distribution was found i) between ICL and OCL and ii) according to the colonic level and NANC examined. In particular, OCL possessed both choline@ nerves, except for VIPergic ones at CC], and ICL had none of them at CCJ and was lacking in nitride@ and NPYergic nerves at the other colonic levels. In conclusion, these data indicate that JCL differs from OCL and CCJ from the other colonic levels in both cell population and innervation and are a further proof in favour that ICL and CCJ play specific roles in colonic motility.lt remains, however, to demonstrate whether or not the region-specific innervation we found - absence at the ICL all along the entire colon, and even more at CCJ, of some of the neurotransmitters present at OCL - is in contrast with the presumptive roles played by ICL pacemaker - and CCJ -sphincterial activity. This work was supported
by a 60% MURST grant.
COORDINATE REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE(iNOS) AND SUPEROXIDE DISMUTASE (SOD) IN MYENTERIC NEURONS BY INFLAMMATION: POTENTIAL NEUROPROTECTIVE ROLE FOR SOD. CL Tannahill, JF Valentine, SA Stevenot, HS GI Med & Nick, JE Sallustio, EY Eaker. Biochem & Mol Bio, U of Fl, Gainesville, FL. Nitric oxide (NO) acts as an inhibitory enteric nervous neurotransmitter in the system and is generated via a constitutive In macrophages and intestinal NOS (cNOS) epithelial cells in the inflammatory state, inducible NOS (iNOS) NO can be formed via NO can react with oxygen radicals to LUSO, peroxynitrite, which can cause form We have reported that ytotoxicity. superoxide dismutase ntestinal manganese removal of o,which catalyses (MnSOD), radicals to protect the cell, is induced by &nflammation and cytokines. We now examine in rats the expression of MnSOD and iNOS in ryenteric plexus neurons (MP)in acetic acid nduced colitis and in isolated myenteric heurons in culture treated with cytokines ILpp and TNF. MnSOD staining was studied with polyclonal antibody and total NOS with the Expression of ADPH diaphorase reaction. evaluated by and iNOS mF.NAs were nSOD resulted in a Northern analysis. Colitis dramatic increase in SOD staining and mRNA for both SOD and iNOS in dissected MP and mucosa. Cultured MP exposed to IL-1p and TNF demonstrated increased staining for SOD and increased SOD mRNA levels. NADPH diaphorase localized NOS to neurons and IL-1p induced a in these dramatic increase in iNOS mRNA cultured cells. Thus, we show iNOS is present in MP which is inducible by inflammation and kytokines and the concomitant induction oF nSOD may play a neuroprotective role.
1229
LOW DOSE CALCIUM CHANNEL BLOCKERS (CCB) EXHIBIT AGONIST EFFECTS ON COLONIC CONTRACTILITY. James Rvan. Leif Nordberg, Henry Parkman, and Joseph Fondacaro. Depts of Physiology and Medicine, Temple Univ. Sch. Medicine, Philadelphia, PA 8 Division of Pharmacology, Marion Merrell Dow, Cincinnati, Ohio. CCB have potent inhibitory effects on GI tract smooth muscle contraction in vitro. In contrast, data from in studies are inconsistent with respect to the ability of CCB to affect GI transit. This is particularly true for the colon, where data are available to suggest that CCB either delay or have no effect on transit. These discrepancies may be related to the dosage and class of channel blocker. The PURPOSE of this study was to compare the effects of three CCB (Diltiazem, Nifedipine, and Verapamil) on spontaneous and acetylcholine (ACh, 1 uM) induced contraction of longitudinal and circular muscle from the rabbit proximal coton. METHODS: Tissues were exposed to the CCB (10.‘*M to lOAM) for 5 min prior to determining their effect on spontaneous and ACh stimulated contractions. RESULTS: 1) At low doses (10.“M to lOaM) each CCB increased the spontaneous contractile activity of each muscle layer, with Diltiazem having the greatest effect. Longitudinal muscle was the most affected by the agonist effects of the CCB. 2) At low doses, Diltiazem and Verapamil (10.“M to 108M) also produced a dose-dependent increase in the contractile response’ to ACh. The effect was most pronounced in the longitudinal layer. 3) Diltiazem was both more potent and more efficacious than Verapamil in augmenting ACh induced contractions. 4) Nifedipine had no effect on ACh induced contractions. 5) At doses greater than 1OaM, each CCB produced dose-dependent inhibition of ACh-induced contractions. CONCLUSIONS: 1) The effect of CCB on colonic contractility is dose dependent, with agonist effects demonstrable at doses less than IO-‘M. 2) The ability to act as an agonist also appears to depend upon the CCB studied. The dose-dependent effects of CCB should be considered when designing in vivo studies. Supported by a grant from MMD Res Institute.
BUNDLES OF INTERSTITIAL CELLS OF CAJAL AND MUSCLE CELLS IN COLONIC SUBMUCOSA: CROSS-COUPLING OF CIRCULAR LAMELLAE? L.Thunebere and J.J.Rumessen. Dept. of Medical Anatomy, Section C, University of Copenhagen, Denmark. &,&g~8& The circular muscle of larger species is divided by radial septa into discrete, circular lamellae. Longitudinal electrical coupling between these lamellae has been registered, and found dependent on an intact submucosal interface’. We have studied the morphological basis for this coupling by electron microscopy. Besllts; Interstitial cells of Caial (ICC) contacted the circular muscle cells, either directly (mouse, guinea pig, rabbit) or indirectly through an interposed layer of transitional, brancbed muscle cells (bSM) (dog, pig, human). In deeper parts of the submucosa discrete, longitudiily/obliquely oriented bundles of 1CC were also present. By electron microscopy, submucosal bundleswere not observedin mousecolon, while partially fibroblast-ensheathed bundles were Present in rabbit and prominent in guinea pig and dog. Bundles were connected to circular muscle and could be followed for several hundred microns across septa. Tbe distribution of nucleated profiles suggested that one type of bundle consisted of single rows of overlapping ICC (guinea pig, rabbit, dog, human). In addition, canine and human colon contained thick bundles of 5-6 overlaP@g bSM, in dog including ICC. Bundles contained small nerves in guinea pig and dog. ICC and bSM were connected-by gap junctions (not observed in human), mtermediate junctions and close appositions. m Our data suggest that ICC/bSM bundles may provide a pathway for longitudinal electrical coupling in colonic circular muscle, in addition to other regulatory roles (pacemaking). ‘Conklin JL, Du C. Pathways of slow-wave propagation in proximal colon of cats. Am J Phys 1990;258: G8944903.
October 1994
NONADRENERGIC-NONCHOLINERGIC (NANC) AND CHOLINERGIC INNERVATION IN THE CIRCULAR MUSCLE STUDY. LAYER OF HUMAN COLON. AN HISTOCHEMICAL P.Matini and M.S.Faussone-Pellegrini. Dept.Hum.Anat. and Histol. University of Florence, Florence, Italy. Two differently arranged portions have been identified in the circular muscle layer of the human colon: a main (outer, CCL) and a submucosal(inner, ICL)portion.ICLhaspeculiarsmoothmusclecells and interstitial cells of Cajal arranged in a continuous layer, which is thicker at the cecocolonic junction (CCJ)than at right and left colon. Myogenic activity seems to origin at the circular muscle and the occurrence of spontaneous spasms have been observed at CCJ. Nerve fibers are present in the two circular muscle layers of the entire colon. The distribution of both NANC and cholinergic nerves either at ICL OT OCL of the entire human colon has been histochemically detected in order to see if these layers possess innervational differences related to their specific roles. Antibodies against Chat (Chemicon), VIP (Amersham), Nitric Oxide synthase (NOs, gift of dr. B.Mayer, University of Graz), NPY and Galanin (Affiniti) have been used; nerve distribution has been revealed by neuronal enolase immunoreactivity (Affiniti). Specimens of CCJ, right and left colon were surgically obtained (Prof.C.Cortesini, Surgery Clinic, University of Florence) from patients operated on for non stenosing colon carcinoma Duke’s A type. Identical distribution and richness in nerve fibers were observed at all areas examined, while differences in neurotransmitters distribution was found i) between ICL and OCL and ii) according to the colonic level and NANC examined. In particular, OCL possessed both choline@ nerves, except for VIPergic ones at CC], and ICL had none of them at CCJ and was lacking in nitride@ and NPYergic nerves at the other colonic levels. In conclusion, these data indicate that JCL differs from OCL and CCJ from the other colonic levels in both cell population and innervation and are a further proof in favour that ICL and CCJ play specific roles in colonic motility.lt remains, however, to demonstrate whether or not the region-specific innervation we found - absence at the ICL all along the entire colon, and even more at CCJ, of some of the neurotransmitters present at OCL - is in contrast with the presumptive roles played by ICL pacemaker - and CCJ -sphincterial activity. This work was supported
by a 60% MURST grant.
COORDINATE REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE(iNOS) AND SUPEROXIDE DISMUTASE (SOD) IN MYENTERIC NEURONS BY INFLAMMATION: POTENTIAL NEUROPROTECTIVE ROLE FOR SOD. CL Tannahill, JF Valentine, SA Stevenot, HS GI Med & Nick, JE Sallustio, EY Eaker. Biochem & Mol Bio, U of Fl, Gainesville, FL. Nitric oxide (NO) acts as an inhibitory enteric nervous neurotransmitter in the system and is generated via a constitutive In macrophages and intestinal NOS (cNOS) epithelial cells in the inflammatory state, inducible NOS (iNOS) NO can be formed via NO can react with oxygen radicals to LUSO, peroxynitrite, which can cause form We have reported that ytotoxicity. superoxide dismutase ntestinal manganese removal of o,which catalyses (MnSOD), radicals to protect the cell, is induced by &nflammation and cytokines. We now examine in rats the expression of MnSOD and iNOS in ryenteric plexus neurons (MP)in acetic acid nduced colitis and in isolated myenteric heurons in culture treated with cytokines ILpp and TNF. MnSOD staining was studied with polyclonal antibody and total NOS with the Expression of ADPH diaphorase reaction. evaluated by and iNOS mF.NAs were nSOD resulted in a Northern analysis. Colitis dramatic increase in SOD staining and mRNA for both SOD and iNOS in dissected MP and mucosa. Cultured MP exposed to IL-1p and TNF demonstrated increased staining for SOD and increased SOD mRNA levels. NADPH diaphorase localized NOS to neurons and IL-1p induced a in these dramatic increase in iNOS mRNA cultured cells. Thus, we show iNOS is present in MP which is inducible by inflammation and kytokines and the concomitant induction oF nSOD may play a neuroprotective role.
1229
LOW DOSE CALCIUM CHANNEL BLOCKERS (CCB) EXHIBIT AGONIST EFFECTS ON COLONIC CONTRACTILITY. James Rvan. Leif Nordberg, Henry Parkman, and Joseph Fondacaro. Depts of Physiology and Medicine, Temple Univ. Sch. Medicine, Philadelphia, PA 8 Division of Pharmacology, Marion Merrell Dow, Cincinnati, Ohio. CCB have potent inhibitory effects on GI tract smooth muscle contraction in vitro. In contrast, data from in studies are inconsistent with respect to the ability of CCB to affect GI transit. This is particularly true for the colon, where data are available to suggest that CCB either delay or have no effect on transit. These discrepancies may be related to the dosage and class of channel blocker. The PURPOSE of this study was to compare the effects of three CCB (Diltiazem, Nifedipine, and Verapamil) on spontaneous and acetylcholine (ACh, 1 uM) induced contraction of longitudinal and circular muscle from the rabbit proximal coton. METHODS: Tissues were exposed to the CCB (10.‘*M to lOAM) for 5 min prior to determining their effect on spontaneous and ACh stimulated contractions. RESULTS: 1) At low doses (10.“M to lOaM) each CCB increased the spontaneous contractile activity of each muscle layer, with Diltiazem having the greatest effect. Longitudinal muscle was the most affected by the agonist effects of the CCB. 2) At low doses, Diltiazem and Verapamil (10.“M to 108M) also produced a dose-dependent increase in the contractile response’ to ACh. The effect was most pronounced in the longitudinal layer. 3) Diltiazem was both more potent and more efficacious than Verapamil in augmenting ACh induced contractions. 4) Nifedipine had no effect on ACh induced contractions. 5) At doses greater than 1OaM, each CCB produced dose-dependent inhibition of ACh-induced contractions. CONCLUSIONS: 1) The effect of CCB on colonic contractility is dose dependent, with agonist effects demonstrable at doses less than IO-‘M. 2) The ability to act as an agonist also appears to depend upon the CCB studied. The dose-dependent effects of CCB should be considered when designing in vivo studies. Supported by a grant from MMD Res Institute.
BUNDLES OF INTERSTITIAL CELLS OF CAJAL AND MUSCLE CELLS IN COLONIC SUBMUCOSA: CROSS-COUPLING OF CIRCULAR LAMELLAE? L.Thunebere and J.J.Rumessen. Dept. of Medical Anatomy, Section C, University of Copenhagen, Denmark. &,&g~8& The circular muscle of larger species is divided by radial septa into discrete, circular lamellae. Longitudinal electrical coupling between these lamellae has been registered, and found dependent on an intact submucosal interface’. We have studied the morphological basis for this coupling by electron microscopy. Besllts; Interstitial cells of Caial (ICC) contacted the circular muscle cells, either directly (mouse, guinea pig, rabbit) or indirectly through an interposed layer of transitional, brancbed muscle cells (bSM) (dog, pig, human). In deeper parts of the submucosa discrete, longitudiily/obliquely oriented bundles of 1CC were also present. By electron microscopy, submucosal bundleswere not observedin mousecolon, while partially fibroblast-ensheathed bundles were Present in rabbit and prominent in guinea pig and dog. Bundles were connected to circular muscle and could be followed for several hundred microns across septa. Tbe distribution of nucleated profiles suggested that one type of bundle consisted of single rows of overlapping ICC (guinea pig, rabbit, dog, human). In addition, canine and human colon contained thick bundles of 5-6 overlaP@g bSM, in dog including ICC. Bundles contained small nerves in guinea pig and dog. ICC and bSM were connected-by gap junctions (not observed in human), mtermediate junctions and close appositions. m Our data suggest that ICC/bSM bundles may provide a pathway for longitudinal electrical coupling in colonic circular muscle, in addition to other regulatory roles (pacemaking). ‘Conklin JL, Du C. Pathways of slow-wave propagation in proximal colon of cats. Am J Phys 1990;258: G8944903.