Shortening velocity is different in longitudinal and circular muscle layers of the rabbit urethra

The aim of the study was to investigate whether the functional difference between circular and longitudinal muscles in the female rabbit urethra is reflected in their shortening properties and lactate dehydrogenase (LDH) activity. For mechanical experiments the preparations were chemically skinned to avoid influence of membrane-related mechanisms and to enable maximal activation. Force velocity relations and the maximal shortening velocity (v(max)) were determined using the isotonic quick-release method. The v(max) was three times higher in longitudinal muscle. LDH isoform pattern was determined on agarose gels. The M-subunit, favourable for lactate formation, constituted 70% of the total in both types of muscle. There was no difference in the LDH isoform pattern despite the marked difference in v(max). We conclude that the difference in v(max) reflects differences in the contractile machinery itself. These mechanical characteristics are advantageous for the role of the circular as a tonic muscle contracting during bladder filling, and the longitudinal as a phasic muscle active in opening up the urethra during micturition.     

Alpha 2-adrenoceptors in the guinea-pig uterus: heterogeneity in the circular and longitudinal smooth muscle layers

Experiments have been performed to determine whether the antisecretory (antidiarrhoeal) actions of difenoxin and loperamide are mediated by enteric neurones. An iso-osmotic perfusion solution was circulated around the lumen of the jejunum of anaesthetised rats. Vasoactive intestinal peptide was infused intra-arterially to induce net fluid secretion which was inhibited by difenoxin (ED50, 0.23 mg/kg) and loperamide (ED50, 0.5 mg/kg). However, neither were able to restore the fluid transport rate to the control level of absorption. The antisecretory effects of difenoxin (0.77 mg/kg) and loperamide (0.6 mg/kg) were blocked by the opiate receptor antagonist naloxone (2 mg/kg). Their effects were also abolished by pretreatment with the 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA; 200 mg/kg; with desmethylimipramine given beforehand to protect noradrenergic nerves and enhance 5-HT depletion). The effect of difenoxin was blocked with methiothepin (1 mg/kg) and methysergide (30 micrograms/kg) but not ketanserin (30 micrograms/kg), ritanserin (30 mg/kg), ondansetron (10 micrograms/kg) or ICS 205-930 (3 mg/kg). None of the above 5-HT receptor antagonists modified the antisecretory effect of loperamide. The antisecretory effect of difenoxin but not loperamide was prevented by phentolamine (2 mg/kg) and by pretreatment with 6-hydroxy-dopamine (150 mg/kg total). It is concluded that both difenoxin and loperamide inhibit net fluid secretion by indirect mechanisms. It is proposed that the initial action is on enteric mu-opiate receptors and that this results in the release of 5-HT. In the case of difenoxin, the 5-HT may act on 5-HT1-like receptors to release noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.     

Characterization of cholecystokinin receptors in canine intestinal circular muscle

We localized and characterized the binding of [3H](+/-)-L364,718 in canine small intestine circular muscle. The highest densities of [3H]L364,718 binding were located in the fraction enriched in deep muscular plexus synaptosomal membranes. In this fraction [3H]L364,718 binding was of high density (Bmax 136.78 +/- 53.66 fmol/mg) and high affinity (Kd 1.67 +/- 0.74 nM). Kinetics studies revealed that binding was reversible and yielded a similar Kd value. L364,718, CCK-8-S, and L365,260 fully displaced [3H]L364,718 binding, but ligands at CCKB receptors, gastrin-17, and YM022 did not. Therefore, CCKA receptors in canine intestine circular muscle are located on nerve endings.     

Interstitial cells of Cajal as targets for pharmacological intervention in gastrointestinal motor disorders

Interstitial cells of Cajal (ICCs) have recently been identified as the pacemaker cells for contractile activity of the gastrointestinal tract. These cells generate the electrical 'slow-wave' activity that determines the characteristic frequency of phasic contractions of the stomach, intestine and colon. Slow waves also determine the direction and velocity of propagation of peristaltic activity, in concert with the enteric nervous system. Characterization of receptors and ion channels in the ICC membrane is under way, and manipulation of slow-wave activity markedly alters movement of contents through the gut organs. Here Jan Huizinga, Lars Thuneberg, Jean-Marie Vanderwinden and Jüri Rumessen, suggest that, as ICCs are unique to the gut, they might be ideal targets for pharmacological intervention in gastrointestinal motility disorders, which are very common and costly.     

Interstitial cells of Cajal mediate enteric inhibitory neurotransmission in the lower esophageal and pyloric sphincters

BACKGROUND & AIMS: Previous studies have suggested that a specific class of interstitial cells of Cajal (ICC) act as mediators in nitrergic inhibitory neurotransmission. The aim of this investigation was to examine the role of intramuscular ICC (IC-IM) in neurotransmission in the murine lower esophageal (LES) and pyloric sphincters (PS). METHODS: Immunohistochemistry and electrophysiology were used to study the distribution and role of IC-IM. RESULTS: The LES and PS contain spindle-shaped IC-IM, which form close relationships with nitric oxide synthase-containing nerve fibers. The PS contains ICC within the myenteric plexus and c-Kit immunopositive cells along the submucosal surface of the circular muscle. IC-IM were absent in the LES and PS of c-kit (W/Wv) mutant mice. Using these mutants, we tested whether IC-IM mediate neural inputs in the LES and PS. Although the distribution of inhibitory nerves was normal in W/Wv animals, NO-dependent inhibitory neurotransmission was reduced. Hyperpolarizations to sodium nitroprusside were also attenuated in W/Wv animals. CONCLUSIONS: The data suggest that IC-IM play an important role in NO-dependent neurotransmission in the LES and PS. IC-IM may be the effectors that transduce NO signals into hyperpolarizing responses. Loss of IC-IM may interfere with relaxations and normal motility in these sphincters.     

Intercellular communication between dorsal root ganglion cells and colonic smooth muscle cells in vitro

This study demonstrates that the use of high field 1H NMR spectroscopy permits individual detection of phosphatidylcholine and sphingomyelin molecules at the surface of native low density lipoprotein (LDL) particles. Distinct behaviour was observed for the choline head group -N(CH3)3 resonances of these different phospholipids revealing preferential immobilisation for phosphatidylcholine. This suggests the existence of reversible and irreversible phosphatidylcholine-apolipoprotein B interactions and is consistent with microdomain formation at the surface monolayer of LDL. The novel resonance assignment and results show that 1H NMR can provide efficient and practical means for future studies on the structure and dynamics at the LDL surface.     

Carbohydrate sulfotransferases: mediators of extracellular communication

Sulfated carbohydrates mediate diverse extracellular recognition events in both normal and pathological processes. The sulfotransferases that generate specific carbohydrate 'sulfoforms' have recently been recognized as key modulators of these processes and therefore represent potential therapeutic targets.     

The neurochemical coding and projections of circular muscle motor neurons in the human colon

BACKGROUND & AIMS: Enteric neurons can be characterized by their chemical coding, projections, and morphology. The aim of this study was to describe the different classes of human colonic circular muscle motor neurons. METHODS: Human colonic circular muscle motor neurons were identified by retrograde tracing with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (Dil) applied to the circular muscle layer. Whole-mount preparations of the myenteric plexus were then double-labeled with antisera to choline acetyltransferase (ChAT) and/or nitric oxide synthase (NOS), or NOS and vasoactive intestinal peptide (VIP), and the position and immunoreactivity of Dil-filled neurons were recorded. RESULTS: Fifty-two percent of all Dil-filled neurons were ChAT immunoreactive, and 86% of these projected up to 11 mm orally, with 14% projecting short distances anally. Forty-eight percent of the Dil-filled neurons were NOS immunoreactive, and 77% of these projected up to 19 mm anally, with 23% projecting no more than 6 mm orally. A subpopulation of these NOS-immunoreactive motor neurons were also VIP-immunoreactive. A small population of myenteric neurons was immunoreactive for both ChAT and NOS, but none projected to the circular muscle. NOS-immunoreactive motor neurons projected for longer distances than those with ChAT immunoreactivity and were larger. CONCLUSIONS: There are two classes of human colonic motor neurons: one is excitatory (ChAT-immunoreactive) and mainly projects orally and the other is inhibitory (NOS +/- VIP immunoreactive) and projects preferentially anally.     

Spontaneous and evoked inhibitory junction potentials in the circular muscle layer of mouse colon

Intracellular microelectrodes were used to record spontaneous and evoked inhibitory junction potentials (IJPs) from the circular muscle layer of an in vitro preparation of whole mouse colon. Membrane potential recordings were made from cells of the mid to distal region of colon at 36 +/- 1 degrees C in a modified Krebs' solution that contained atropine (1 microM) and nifedipine (1-2 microM). Spontaneously occurring hyperpolarisations of irregular amplitude and frequency (range: up to 20 mV and 2 Hz) were recorded that were resistant to N(G)-nitro-L-arginine (NOLA, 100 microM), but were abolished by tetrodotoxin (TTX, 1.6 microM) or apamin (250 nM). These were considered to be spontaneous IJPs as a consequence of activity in inhibitory motor neurons. Single electrical stimuli (0.6 ms, 15 V), elicited a fast IJP, whose time course could be superimposed on spontaneous IJPs of similar amplitude. The amplitude of evoked IJPs was not depressed by NOLA (100 microM). However, in NOLA (100 microM), further addition of apamin (250 nM) significantly depressed the amplitudes of the evoked IJPs by 44%. NOLA- and apamin-resistant evoked IJPs were abolished by TTX (1.6 microM). It is suggested, that in the circular muscle layer of mouse colon, NO does not mediate the fast hyperpolarisations associated with spontaneous or evoked IJPs. Apamin abolished spontaneous IJPs, but electrical stimuli evoked an IJP with apamin-sensitive and resistant components both of which were non-nitrergic in origin.     

Association between pelvic muscle mass and canine hip dysplasia

OBJECTIVE: To investigate the relationship between pelvic muscle mass and development and expression of canine hip dysplasia (CHD). DESIGN: Prospective study. ANIMALS: 5 Greyhounds with anatomically normal hip joints, 59 German Shepherd Dogs (23 with CHD, 24 with near-normal hip joints, and 12 with normal hip joints), and 18 German Shepherd Dog-Greyhound crossbreeds (7 with CHD, 6 with near-normal hip joints, and 5 with normal hip joints) between 12 and 47 months old in which pelvic muscle mass was evaluated. Pectineal muscle and hip joint development were evaluated in 25 German Shepherd Dogs at 8 and 16 or 24 weeks of age. PROCEDURES: For evaluation of pelvic muscle mass, individual pelvic muscles were weighed and hip joints were assigned a score on the basis of severity of degenerative changes. For evaluation of pectineal muscle development, muscle sections were stained and examined. RESULTS: Pelvic muscle mass was greatest in Greyhounds, intermediate in crossbred dogs, and smallest in German Shepherd Dogs. Differences in pelvic muscle mass among breeds were attributable to differences in weights of individual muscles. Hip score was negatively correlated with pelvic muscle mass and weights of selected pelvic muscles. Dogs with pectineal hypotrophy at 8 weeks of age had type-2 muscle fiber paucity or muscle fiber-type grouping at 16 or 24 weeks of age. At 8 weeks of age, hip joints were composed of multiple centers of ossification, and the acetabulum was largely cartilaginous. By 24 weeks of age, the pelvic bones were largely, although incompletely, fused. CLINICAL IMPLICATIONS: Diminished pelvic muscle mass in dogs with CHD and altered muscle fiber size and composition in 8-week-old dogs that subsequently develop CHD strongly suggest that abnormalities of pelvic musculature are associated with development of CHD. The complex development of the hip joint from multiple centers of ossification may make the joint susceptible to abnormal modeling forces that would result from abnormalities in pelvic muscle mass.     

Tachykinins mediate noncholinergic excitatory neural responses in the circular muscle of rat proximal colon

Using the sucrose-gap technique, we attempted to assess a role for tachykinins (TKs) in mediating noncholinergic excitatory junction potential (EJP) and contraction, in the circular muscle of rat proximal colon. Excitatory responses were evoked by submaximal electrical field stimulation (EFS) in the presence of atropine (1 microM), guanethidine (1 microM), indomethacin (10 microM), and N(omega)-nitro-L-arginine methyl ester (L-NAME) (100 microM). The NK1 receptor antagonist, SR 140,333 (up to 3 microM) or the NK2 receptor antagonists, SR 48,968 and MEN 10,627 (up to 5 microM) produced a partial inhibition of the excitatory responses to EFS. The co-administration of the selective NK1 and NK2 receptor antagonists produced additive effects on the responses to EFS. Selective NK1 receptor agonist, [Sar9, Met (O2)11]-substance P, induced depolarization and contraction, antagonized by SR 140,333, but not by NK2 receptor antagonists. NK2 receptor agonist, [betaAla8]-neurokinin A (4-10), also produced electrical and mechanical excitatory effects that were antagonized by SR 48,968 or MEN 10,627, but not by the NK1 receptor antagonist. Our results provide evidence that, in circular muscle of rat colon, endogenous tachykinins are the main excitatory transmitters for nonadrenergic, noncholinergic (NANC) excitation and their action is mediated by both NK1 and NK2 receptors.     

Bilirubin levels in subarachnoid clot and effects on canine arterial smooth muscle cells

BACKGROUND AND PURPOSE: Previous studies have suggested that bilirubin is a potential contributor to cerebral vasospasm. The purpose of this investigation was to determine whether bilirubin accrues in subarachnoid clot, whether its vasoconstrictive effect could involve a direct action on arterial smooth muscle cells, and, if so, whether bilirubin affects their Ca2+ uptake. METHODS: Subarachnoid clots were analyzed for bilirubin using high-performance liquid chromatography. The length and 45Ca2+ uptake of vascular smooth muscle cells enzymatically dissociated from canine carotid arteries were measured before and after exposure to bilirubin solution. Additional experiments were conducted on cultured smooth muscle cells from canine basilar artery and on ATP-depleted cardiac myocytes. RESULTS: Mean +/- SE bilirubin concentration in experimental clot was 263 +/- 35.7 mumol/L. Vascular smooth muscle cells exposed to bilirubin showed progressive shortening (P < .01) and an increased uptake of 45Ca2+ (P < .001). Contraction was prevented by Ca(2+)-free media but not by verapamil. Experiments with heart myocytes showed that bilirubin caused an increased uptake of 45Ca2+ but not of [14C]sucrose. CONCLUSIONS: The results indicate that bilirubin accrues in subarachnoid clot, that it exerts a direct constrictive effect on arterial smooth muscle cells, and that this effect is associated with an increased uptake of Ca2+. Studies on heart myocytes suggest that the Ca2+ uptake induced by bilirubin could be due to a selective increase in membrane permeability to Ca2+.     

Caffeine and ryanodine may act on the plasma membrane of the circular muscle at the flexure region in the guinea-pig colon

The actions of caffeine and ryanodine on the spontaneous rhythmic activities in the guinea-pig colon were studied by mechanical tension recording. Caffeine reduced the amplitude of the spontaneous rhythmic activity at low concentration (0.3 mM-1 mM). At high concentration (3-10 mM), it induced a phasic transient contraction. The spontaneous rhythmic activity and a phasic contraction induced by caffeine, were blocked by verapamil (3 microM) or by removal of external Ca2+. Ryanodine affected neither resting tension nor frequency of spontaneous activity at 1 microM. However in the circular muscle strips pretreated with ryanodine, a sustained contraction was initiated after the removal of caffeine (10 mM). Continuous Ca2+ influx was necessary for spontaneous rhythmic activities and a phasic transient contraction, because it was abolished completely by the removal of external Ca2+, Verapamil (3 microM), a voltage gated L-type Ca2+ channels blocker, inhibited the spontaneous rhythmic activities and also inhibited phasic transit contraction followed by a sustained contraction induced by 10 mM caffeine. Our results suggest that caffeine may produce a sustained contraction by activating verapamil sensitive Ca2+ channel. In the muscle pretreated with both caffeine and ryanodine, continuous Ca2+ influx may occur also through verapamil sensitive pathway.     

Tachykinin NK1 but not NK2 receptors mediate non-cholinergic excitatory junction potentials in the circular muscle of guinea-pig colon

1. The effect of tachykinin NK1 and NK2 receptor antagonists on noncholinergic excitatory junction potentials (e.j.ps) evoked by electric field stimulation (EFS) in the circular muscle of the guinea-pig proximal colon was investigated by means of a sucrose-gap technique. 2. In the presence of 1 microM atropine, submaximal EFS (10 Hz, 20-30 V, 0.5 ms pulse width, 1 s train duration) evoked an inhibitory junction potential (i.j.p.) followed by e.j.p. with superimposed action potentials (APs) and contraction. Addition of either NG-nitro-L-arginine (L-NOARG, 0.1 mM) or apamin (0.1 microM) inhibited the evoked i.j.p. and the combined administration of the two agents almost abolished it. In the presence of both L-NOARG and apamin, an atropine-resistant e.j.p. was the only electrical response evoked by EFS in 50% of cases and a small i.j.p. (10% of original amplitude) followed by e.j.p. was evident in the remainder. 3. In the presence of L-NOARG and apamin, the tachykinin NK1 receptor antagonists, (+/-)-CP 96,345 and GR 82,334 (10 nM-3 microM) concentration-dependently inhibited the atropine-resistant e.j.p. and accompanying contraction evoked by EFS. EC50 values were: 0.77 microM (e.j.p. inhibition) and 0.22 microM (inhibition of contraction) for (+/-)-CP 96,345; 0.61 microM (e.j.p. inhibition) and 0.20 microM (inhibition of contraction) for GR 82,334. The tachykinin NK2 receptor antagonists, MEN 10,376 (up to 3 microM) and SR 48,968 (up to 1 microM) had no effect on the atropine-resistant e.j.p. MEN 10,376 (3 microM) but not SR 48,968 produced a slight inhibition of the evoked contraction. 4. (+/- )-CP 96,345 (3 microM) and GR 82,334 (3 microM) markedly reduced (81 and 89% inhibition, respectively)the atropine-resistant ej.p. in the absence of L-NOARG and apamin, without affecting the ij.p. MEN 10,376 (3 microM) and SR 48,968 (1 microM) had no significant effect on noncholinergic ij.p. and ej.p. evoked in the absence of apamin and L-NOARG.5. The electrical and mechanical responses to the NK, receptor agonist [Sar9]substance P (SP) sulfone were blocked by (+/-)-CP 96,345 (3 1M) or GR 82,334 (3 microM) which, at the same concentration, failed to affect the responses to the NK2 receptor agonist [PAla8] neurokinin A (NKA) (4-10). In contrast, MEN10,376 (3 microM) or SR 48,968 (1 microM) blocked the response to [beta Ala8]NKA(4-10) without affecting the response to [Sar9]SP sulfone.6. In the presence of L-NOARG and apamin, and in the absence of atropine, EFS of low pulse width(0.02-0.03 ms, other parameters as above) produced cholinergic ej.ps and contraction which were unaffected by GR 82,334 (3 microM). (+/-)-CP 96,345 (3 JAM) produced 24% reduction in the area of the atropine-sensitive ej.p. without affecting the peak amplitude of ej.p. or contraction.7. These findings demonstrate that the noncholinergic ej.ps and accompanying contraction of the circular muscle of the guinea-pig colon are produced through activation of intramural tachykininergic nerves and that the resultant smooth muscle response is almost entirely mediated through NK1 receptors.     

Direct inhibitory effect of adrenomedullin and guanylin on isolated caecal circular smooth muscle cells of guinea pig

Smooth muscle cells isolated from the caecal circular smooth muscle layers of the guinea pig were used to determine whether adrenomedullin and guanylin can inhibit the contractile response produced by 10(-9) M cholecystokinin octapeptide (CCK-8). In addition, to elucidate each intracellular mechanisms, we examined the effects of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate guanylate cyclase, and an inhibitor of soluble guanylate cyclase on the adrenomedullin- or guanylin-induced relaxation of the caecal circular smooth muscle cells. Both adrenomedullin and guanylin inhibited the contractile response produced by CCK-8 in a dose-dependent manner, with IC50 values of 0.12 nM and 2.4 pM, respectively. An inhibitor of cAMP-dependent protein kinase significantly inhibited the relaxation produced by adrenomedullin. In contrast, an inhibitor of particulate guanylate cyclase and an inhibitor of soluble guanylate cyclase did not have any significant effect on the relaxation produced by adrenomedullin. On the other hand, an inhibitor of particulate guanylate cyclase significantly inhibited the guanylin-induced relaxation, although an inhibitor of cAMP-dependent protein kinase and an inhibitor of soluble guanylate cyclase did not have any significant effect on the guanylin-induced relaxation. In this study, we first demonstrated the direct inhibitory effects of adrenomedullin via cAMP system and guanylin via particulate guanylate cyclase system on the isolated caecal circular smooth muscle cells.     

Influence of Shenqing Recipe on Morphology and Quantity of Colonic Interstitial Cells of Cajal in Trinitrobenzene Sulfonic Acid Induced Rat Colitis

Objective To observe the influence of Shenqing Recipe (SQR),a kind of Traditional Chinese Medicine,on the morphology and quantity of colonic interstitial cells of Cajal (ICC) in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis,and to investigate the possible mechanism of SQR in regulating intestinal dynamics.Methods Sixty rats were randomly divided into normal control,model I ,model Ⅱ,mesalazine,and high-dose,and low-dose SQR groups with 10 rats in each group.TNBS (10 mg) dissolved in 50% etha-nol was instilled into the lumen of the rat colon of the latter five groups to induce colitis.On the 4th day after administration of TNBS,each treatment group was administered one of the following formulations by enteroclysis gavage once a day for 7 days:600 mg · kg-1·d-1 mesalazine,2.4 g·kg-1·d-1 SQR,and 1.2 g·kg-1·d-1 SQR.Model Ⅱ rats received normal saline solution.After 7 days colonic samples were collected.While the colonic samples of model I group were collected on the 3rd day after TNBS administered.Ultrastructure of ICC in the damaged colonic tissues was observed with transmission electron microscope.Expression of c-kit protein in colonic tissue was determined by immunohistochemical staining and Western blot.Results The ultrastructure of colonic ICC in the rat model of TNBS-induced colitis showed a severe injury,and administration of SQR or mesalazine reduced the severity of injury.Similarly,the expression of c-kit protein of TNBS-induced colitis rat model was significantly decreased compared with the normal control group (P<0.05).Treatment with SQR or mesalazine significantly increased die expression of c-kit protein compared with the administration of control formulations (P<0.05),especially the high-dose SQR group.Conclusion SQR could alleviate and repair the injured ICC,and improve its quantity,which might be involved in regulating intestinal motility.