Muscle Of Guinea-pig Small Intestine Research Articles

Control of non-adrenergic non-cholinergic reflex motor responses in circular muscle of guinea-pig small intestine by Met-enkephalin.

1 A triple organ bath method allowing the synchronous recording of the motor activity of the circular muscle layer belonging to the oral and anal segments of guinea-pig small intestine adjacent to an electrically stimulated middle segment was developed to study the ascending and descending reflex motor responses. 2 Electrical field stimulation (0.8 ms, 40 V, 5 Hz, 10 s) applied to the middle part of the segments elicited tetrodotoxin (1 microm)-sensitive ascending and descending contractile responses of the nonstimulated parts, oral and anal, respectively. The ascending contraction was more pronounced as compared with the descending contraction. 3 In the presence of phentolamine (5 microm), propranolol (5 microm) and atropine (3 microm) a significant decrease in the amplitude of the ascending contraction was seen and a descending relaxation, instead of a contraction was observed. 4 Met-enkephalin applied at a single concentration (0.1 microm) or cumulatively (0.001-1 microm) inhibited both non-adrenergic non-cholinergic (NANC) descending relaxation and ascending contraction with similar efficacy but different potency, IC50 being 5.9 +/- 0.3 and 39.0 +/- 4 nm, respectively. Naloxone (0.5 microm) prevented the effects of Met-enkephalin. 5 L-NNA (0.5 mm), an inhibitor of nitric oxide synthesis, increased the ascending contraction and strongly reduced but not abolished the descending relaxation. l-Arginine (0.5 mm) restored the motor responses to the initial level in l-NNA-pretreated preparations, d-Arginine (0.5 nm) had no effects. 6 Met-enkephalin (0.1 microm) depressed the l-NNA-dependent increase of the ascending contraction and failed to change the l-NNA-resistant part of the descending relaxation. 7 Met-enkephalin did not alter spontaneous NANC mechanical activity. SNP (1 or 10 microm), an exogenous donor of nitric oxide, caused a concentration-dependent relaxation. The effects of SNP persisted in Met-enkephalin (0.1 microm)-pretreated preparations. 8 NANC reflex ascending contraction and descending relaxation were synchronously induced by a local nerve stimulation indicating a functional coactivation of NANC orally projected excitatory and anally directed inhibitory pathways. Acting prejunctionally, Met-enkephalin provided a negative controlling mechanism inhibiting both ascending and descending, mainly nitric oxide mediated, reflex responses. A higher sensitivity of the descending relaxation to Met-enkephalin was observed suggesting an essential role of opioid(s) in reducing the efficacy of descending motor activity.

Clostridium difficile toxin B inhibits carbachol‐induced force and myosin light chain phosphorylation in guinea‐pig smooth muscle: role of Rho proteins

1. Clostridium difficile toxin B glucosylates the Ras-related low molecular mass GTPases of the Rho subfamily thereby inactivating them. In the present report, toxin B was applied as a tool to test whether Rho proteins participate in the carbachol-induced increase in the Ca2+ sensitivity of force and myosin light chain (MLC) phosphorylation in intact intestinal smooth muscle. 2. Small strips of the longitudinal muscle of guinea-pig small intestine were incubated in toxin B (40 ng ml-1) overnight. Carbachol-induced force and intracellular [Ca2+], and, in a separate series, force and MLC phosphorylation, were determined. 3. Carbachol induced a biphasic contraction: an initial rapid increase in force (peak 1) followed by a partial relaxation and a second delayed increase in force (peak 2). The peak of the Ca2+ signal measured with fura-2 preceded peak 1 of force and then declined to a lower suprabasal steady-state level. Peak 2 was not associated with a significant increase in [Ca2+]. Toxin B nearly completely inhibited peak 2 while peak 1 was not significantly inhibited. Toxin B had no effect on the Ca2+ transient. 4. In control strips, MLC phosphorylation at peak 2 was 27.7% which was significantly higher than the resting value (18.6%). The inhibition of the second, delayed, rise in force induced by toxin B was associated with complete inhibition of the increase in MLC phosphorylation. The resting MLC phosphorylation was not significantly different from that of the control strips. 5. The initial increase in MLC phosphorylation determined 3 s after exposure to carbachol was 54% in the control strips. Toxin B also inhibited this initial phosphorylation peak despite the fact that the Ca2+ transient and the initial increase in force were not inhibited by toxin B. This suggests that Rho proteins play an important role in setting the balance between MLC phosphorylation and dephosphorylation reactions even at high levels of intracellular Ca2+. 6. These findings are consistent with the hypothesis that the delayed rise in force elicited by carbachol is due to an increase in the Ca2+ sensitivity of MLC phosphorylation mediated by Rho proteins.

The role of enteric inhibitory motoneurons in peristalsis in the isolated guinea‐pig small intestine.

1. Peristalsis is a co-ordinated motor behaviour in which an anally propagated contraction of the circular muscle propels intraluminal contents. The role of excitatory motoneurons in peristalsis is well established; however the role of enteric inhibitory motoneurons is unknown. 2. A combination of a nitric oxide synthase inhibitor and apamin, which blocks relaxation of the circular muscle of guinea-pig small intestine mediated by enteric inhibitory motoneurons, was used to investigate the role of inhibitory motoneurons in peristalsis in isolated segments of guinea-pig small intestine. 3. N omega-nitro-L-arginine methyl ester (L-NAME, 400 microM) and N omega-nitro-L-arginine (L-NOArg, 100 microM) significantly reduced the threshold volume required to trigger emptying of the intestine. This effect was reversed by L-arginine (4 mM) and L-arginine alone increased the threshold volume for initiation of peristalsis. Sodium nitroprusside (0.1-10 microM), which generates nitric oxide, also increased the threshold volume. L-NAME, L-NOArg, L-arginine and sodium nitroprusside did not alter the maximal intraluminal pressure generated during emptying. Contraction of the longitudinal muscle during the initial phase of fluid infusion was significantly increased by L-NAME and L-NOArg and reduced by sodium nitroprusside (1 nM to 10 microM). 4. Apamin (0.5 microM) did not significantly alter the threshold volume necessary to initiate peristalsis or contraction of the longitudinal muscle. However, the maximal pressure generated when the intestine was emptying was significantly increased. Furthermore, short segments of circular muscle contracted apparently randomly, before peristaltic emptying was triggered. 5. A combination of L-NAME and apamin completely disrupted peristalsis. Contractions of the circular muscle did not always start at the oral end. Stationary contractions as well as contractions propagating orally and anally were observed. 6. It is concluded that enteric inhibitory motoneurons are crucial for peristalsis to occur. They are important in setting the threshold at which peristaltic emptying is triggered, via nitric oxide. They are essential for the propagation of the circular muscle contraction, via an apamin-sensitive mechanism of transmission. Contraction of the longitudinal muscle during peristalsis is partly inhibited by a nitric oxide-mediated mechanism.

Relation between muscarinic receptor cationic current and internal calcium in guinea-pig jejunal smooth muscle cells.

1. The action of carbachol, which activates muscarinic receptors, was studied in single patch-clamped cells where free internal calcium concentration in the cell (Cai2+) was estimated using the emission from the dye Indo-1. Cells were dialysed with potassium-free caesium solution to block any Ca(2+)-activated K(+)-current. 2. Carbachol applied to the cell evoked an initial peak in Cai2+ followed by a smaller sustained rise (plateau) upon which several oscillations in Cai2+ were often superimposed; the changes in inward, cationic current (icarb) followed changes in Cai2+ closely. Calcium entry blocker did not affect these responses. 3. The initial peak in Cai2+ produced by carbachol was due to calcium store release: it was essentially unchanged at +50 mV, and abolished by prior application of caffeine (10 mM) to the cell or by inclusion of heparin (which blocks D-myoinositol 1,4,5-trisphosphate receptors) in the pipette. In contrast, the rise in Cai2+ produced by ATP in rabbit ear artery smooth muscle cells was unaffected by caffeine or heparin as it was due to calcium entry into the cell. 4. The later sustained rise (plateau) in Cai2+ produced by carbachol was due to the entry of calcium into the cell down its electrochemical gradient as it was affected by changing the cell membrane potential or the calcium concentration of the bathing solution. As the sustained rise in Cai2+ produced by caffeine had similar properties, it was suggested that depletion of calcium stores can evoke an increased calcium entry into the cell through some pathway. 5. The cationic current evoked by carbachol was strongly dependent on Cai2+. It was small if any rise in Cai2+ due to calcium store release was prevented by the inclusion of heparin in the pipette solution and increased greatly if calcium entry was provoked through voltage-dependent channels by applying a depolarizing pulse or if calcium was released from stores by caffeine. 6. In the longitudinal muscle of guinea-pig small intestine, activation of muscarinic receptors by carbachol results in the opening of cationic channels; the resulting depolarization increases the frequency of action potential discharge and this determines the degree of contraction. Muscarinic receptor activation opens cationic channels by two mechanisms: release of stored calcium increases Cai2+ and this strongly potentiates a primary mechanism which may involve a G-protein.

Interactions between reflexes evoked by distension and mucosal stimulation: Electrophysiological studies of guinea-pig ileum

Intracellular recording methods were used to examine stereotyped reflexes evoked in the circular muscle of guinea-pig small intestine by distension or repetitive deformation of the mucosal villi, in vitro. Both stimuli evoked compound excitatory junction potentials (EJPs) on the oral side of the site of stimulation and compound inhibitory junction potentials (IJPs) on the anal side. Stimulation of the mucosa by application of 10 μl of 0.5 M HCl evoked similar reflex responses in the circular muscle. The compound EJPs evoked by mucosal stimulation were depressed, but not abolished, by 1 μM hyoscine, indicating that these responses were partially mediated by release of acetylcholine, as are the equivalent responses evoked by distension. The compound EJPs and the compound IJPs evoked by maintained distension or by repeated mechanical stimulation of the mucosa were transient, lasting in most cases for 3–5 s before the membrane potential returned to resting level. This decline (rundown) occurred in part of the circuit close to the site of stimulation as stimuli applied elsewhere during the period of rundown evoked normal EJPs and IJPs. Mechanical stimuli (brush strokes that deformed the mucosal villi) applied to the mucosa at the site of a maintained distension evoked responses of normal amplitude during the period when the response to the distension had declined to zero. In contrast, during the period when the responses to repetitive mechanical stimulation of the mucosa had disappeared, the reflex responses evoked by distension at the same site were substantially augmented. Chemical stimulation of the mucosa with acid also enhanced the responses to distension. These enhancements were observed even though direct responses to mucosal stimulation could no longer be observed. It is concluded that distension evoked reflexes and reflexes evoked by mucosal stimulation are mediated by separate populations of sensory neurons. The results also indicate that sensory stimulation of the mucosa activates a pathway that interacts with the distension reflex pathway to enhance its activity, but has no maintained direct effect on the circular muscle itself.

Ultrastructural analysis of substance P (SP)-immunoreactive nerve fibers in nyenteric ganglia and circular muscle of guinea-pig small intestine

Ultrastructural analysis of substance P (SP)-immunoreactive nerve fibers in nyenteric ganglia and circular muscle of guinea-pig small intestine