A simple model of pacing in mouse intestine to longitudinal (LM) as well as circular muscle (CM) has been developed. Undissected segments of LM or CM from mouse ileum or jejunum were prepared to record contractions, nerve functions were inhibited, and regular spontaneous contractions were recorded. These had the properties expected of interstitial cells of Cajal (ICC) paced contractions: ileum slower than jejunum, inhibited but not abolished by nicardipine, reduced in frequency by cyclopiazonic acid, abolished by Ca(2+)-free media, and high temperature dependence (Q10 approximately 2.6-3.2). Nicardipine significantly reduced the pacing frequency in LM and CM. Intestinal segments from W/W(V) mice had few irregular contractions in CM but had regular contractions in LM. Other differences were found between LM and CM that suggest that the control of pacing of LM differed from pacing of CM. Moreover, both LM and CM segments in wild-type and W/W(V) and after cyclopiazonic acid responded to electrical pacing (50 V/cm, 50 or 100 ms) at 1 pulse per second. Temperature <26 degrees C inhibited electrically paced contractions in CM. These findings suggest that the current models of ICC pacing need to be modified to apply to intact segments of mouse intestine.

Pacing of mouse intestine is driven by spontaneous activity of a network of interstitial cells of Cajal in the myenteric plexus (ICC-MP). So far, highly dissected circular muscle (CM) strips from control and mutant mice lacking ICC-MP and isolated, cultured ICC from newborn control mice were used to analyze its properties. Using intact circular and longitudinal segments of intestine, we recently reported that there were both significant similarities and differences between pacing studied in segments and from isolated, dissected tissues. Here, we report additional similarities and differences in our model from those in highly reduced systems. Similar to cultured or dissected intestine, blockade of sarcoplasmicendoplasmic reticulum Ca 2ϩ pumps with thapsigargin or cyclopiazonic acid reduced pacing frequency, but thapsigargin was less effective than in isolated, cultured ICC. Moreover, inhibition of inositol 1,4,5-trisphosphate (IP3) receptors with xestospongin C, a putative inhibitor of IP3 receptors, failed to affect pacing but successfully blocked increased pacing frequency by phorbol ester. 2-Aminoethoxy-diphenylborate, a putative blocker of IP 3-mediated calcium release, caused a significant decrease in the amplitude and frequency of contractions. The mitochondrial uncoupler carbonyl cyanide ptrifluormethoxyphenylhydrazone blocked pacing and KCl-induced contractions at a concentration of 1 M. The cyclic nucleotide agonists sodium nitroprusside (SNP), forskolin, and 8-bromo-cGMP inhibited pacing in CM. In longitudinal muscle (LM), SNP and forskolin had little effect on pacing. Furthermore, dibutyryl-cAMP did not affect pacing in CM or LM. These results suggest that pacing in intact intestine is under partly similar regulatory control as in more reduced systems. However, pacing in intact intestine is not affected by xestospongin C, which abolishes pacing in isolated, cultured ICC and exhibits attenuated responses to thapsigargin. Also, major differences between LM and CM suggest a separate pacemaker may drive LM.

Pacing of mouse is dependent on the spontaneous activity of interstitial cells of Cajal in the myenteric plexus (ICC-MP). These ICC, as well as intestinal smooth muscle, contain small membrane invaginations called caveolae. Caveolae are signaling centers formed by insertions of caveolin proteins in the inner aspect of the plasma membrane. Caveolins bind signaling proteins and thereby negatively modulate their signaling. We disrupted caveolae by treating intestinal segments with methyl beta-clodextrin (CD) to remove cholesterol or with water-soluble cholesterol (WSC) to load cholesterol. Both of these treatments reduced pacing frequencies, and these effects were reversed by the other agent. These treatments also inhibited paced contractions, but complete reversal was not observed. To evaluate the specificity of the effects of CD and WSC, additional studies were made of their effects on responses to carbamoyl choline and to stimulation of cholinergic nerves. Neither of these treatments affected these sets of responses compared with their respective time controls. Immunochemical and ultrastructural studies showed that caveolin 1 was present in smooth muscle membranes and ICC-MP. CD depleted both caveolin 1 and caveolae, whereas WSC increased the amount of caveolin 1 immunoreactivity and altered its distribution but failed to increase the number of caveolae. The effects of each agent were reversed in major part by the other. We conclude that signaling through caveolae may play a role in pacing by ICC but does not affect responses to acetylcholine from nerves or when added exogenously.

1 Caveolin-1 is a plasma membrane-associated protein that is responsible for caveolae formation. It plays an important role in the regulation of the function of different signaling molecules, among which are the different isoforms of nitric oxide synthase (NOS). 2 Nitric oxide (NO) is known to be an important inhibitory mediator in the mouse gut. Caveolin-1 knockout mice (Cav1 À/À ) were used to examine the effect of caveolin-1 absence on the NO function in the mouse small intestine (ileum and jejunum) compared to their genetic controls and BALB/c controls.3 Immunohistochemical staining showed loss of caveolin-1 and NOS in the jejunal smooth muscles and myenteric plexus interstitial cells of Cajal (ICC) of Cav1 À/À mice; however, nNOS immunoreactive nerves were still present in myenteric ganglia. 4 Under nonadrenergic noncholinergic (NANC) conditions, small intestinal tissues from Cav1 À/À mice relaxed to electrical field stimulation (EFS), as did tissues from control mice. Relaxation of tissues from control mice was markedly reduced by N-omega-nitro-L-arginine (10 À4 M), but relaxation of Cav1 À/À animals was affected much less. Also, Cav1 À/À mice tissues showed reduced relaxation responses to sodium nitroprusside (100 mM) compared to controls; yet there were no significant differences in the relaxation responses to 8-bromoguanosine-3 0 : 5 0 -cyclic monophosphate (100 mM). 5 Apamin (10 À6 M) significantly reduced relaxations to EFS in NANC conditions in Cav1 À/À mice, but not in controls. 6 The data from this study suggest that caveolin-1 gene knockout causes alterations in the smooth muscles and the ICC, leading to an impaired NO function in the mouse small intestine that could possibly be compensated by apamin-sensitive inhibitory mediators.

Varicosities of nitrergic and other nerves end on deep muscular plexus interstitial cells of Cajal or on CD34-positive, c- kit-negative fibroblast-like cells. Both cell types connect to outer circular muscle by gap junctions, which may transmit nerve messages to muscle. We tested the hypotheses that gap junctions transmit pacing messages from interstitial cells of Cajal of the myenteric plexus. Effects of inhibitors of gap junction conductance were studied on paced contractions and nerve transmissions in small segments of circular muscle of mouse intestine. Using electrical field stimulation parameters (50 V/cm, 5 pps, and 0.5 ms) which evoke near maximal responses to nitrergic, cholinergic, and apamin-sensitive nerve stimulation, we isolated inhibitory responses to nitrergic nerves, inhibitory responses to apamin-sensitive nerves and excitatory responses to cholinergic nerves. 18β-Glycyrrhetinic acid (10, 30, and 100 μM), octanol (0.1, 0.3, and 1 mM) and gap peptides (300 μM of40Gap27,43Gap26,37,43Gap27) all failed to abolish neurotransmission. 18β-Glycyrrhetinic acid inhibited frequencies of paced contractions, likely owing to inhibition of l-type Ca2+channels in smooth muscle, but octanol or gap peptides did not. 18β-Glycyrrhetinic acid and octanol, but not gap peptides, reduced the amplitudes of spontaneous and nerve-induced contractions. These reductions paralleled reductions in contractions to exogenous carbachol. Additional experiments with gap peptides in both longitudinal and circular muscle segments after NG-nitro-l-arginine and TTX revealed no effects on pacing frequencies. We conclude that gap junction coupling may not be necessary for pacing or nerve transmission to the circular muscle of the mouse intestine.