Search

The origin and spread of excitation were visualized with fluo 3 fluorescence in tissues isolated from canine gastric antrum. Sheets of circular muscle (5 × 6 mm) had at least 1 (30%) and up to 3 discrete slow-wave pacing sites located near the longitudinal-circular muscle boundary, whereas similarly sized longitudinal sheets had an average of 5 sites (range 3–12 sites) that initiated Ca2+waves. Superimposed fluorescent oscillations (circular muscle) and spikes (longitudinal muscle) were seen to initiate and propagate as distinct events, separate from their underlying activities. Average propagation velocities transverse (6–7 mm/s) and parallel (39–45 mm/s) to the long axis of muscle fibers were similar for each type of event in circular and longitudinal tissues; however, distinct regions where velocities of some (but not all) events decreased by up to an order of magnitude were present. The distance propagated by individual events was limited by collisions with concurrent excitable events or recently activated regions. Complex patterns of excitation in gastrointestinal smooth muscle arise as a result of interactions between multiple pacing sites, heterogeneous conduction velocities, and the interplay of adjacent pacemaker domains.

The motility of the gastrointestinal tract consists of local, non-propulsive mixing (pendular or segmental) and propulsive (peristaltic) movements1,2,3,4,5. It is generally considered that mixing movements are produced by intrinsic pacemakers which generate rhythmic contractions4,5,6, and peristalsis by intrinsic excitatory and inhibitory neural reflex pathways1,2,3,4,5,7,8, but the relationship between mixing and peristalsis is poorly understood4,5,6. Peristalsis is compromised in mice lacking interstitial cells of Cajal9, suggesting that these pacemaker cells10,11,12,13,14 may also be involved in neural reflexes. Here we show that mixing movements within longitudinal muscle result from spontaneously generated waves of elevated internal calcium concentration which originate from discrete locations (pacing sites), spread with anisotropic conduction velocities in all directions, and terminate by colliding with each other or with adjacent neurally suppressed regions. Excitatory neural reflexes control the spread of excitability by inducing new pacing sites and enhancing the overall frequency of pacing, whereas inhibitory reflexes suppress the ability of calcium waves to propagate. We provide evidence that the enteric nervous system organizes mixing movements to generate peristalsis, linking the neural regulation of pacemakers to both types of gut motility.

Microelectrode techniques and the fluorescent Ca2+ indicator indo-1 were used to measure membrane potential, cytosolic Ca2+ ([Ca2+]cyt), and muscle tension simultaneously in canine antral smooth muscles. Responses of muscles from the myenteric and submucosal regions were compared, since electrical activity and excitation-contraction coupling in these regions differ. The upstroke phase of electrical slow waves in both regions induced an increase in [Ca2+]cyt. In myenteric muscles the plateau phase of slow waves often caused either a further rise in [Ca2+]cyt or maintenance of the level reached during the upstroke event. In submucosal muscles, the plateau phase was significantly smaller and did not induce a second phase in the Ca2+ transient. Contractions were related to the amplitudes of Ca2+ transients. Acetylcholine (ACh; 3 x 10(-8)-10(-6) M) increased the amplitude and duration of the plateau phase of slow waves in a concentration-dependent manner. ACh also increased the second phase of Ca2+ transients and contractile responses associated with the plateau potential. In submucosal muscles ACh induced a significant increase in the plateau phase of the slow wave and increased the corresponding phase of Ca2+ transient. Nicardipine (10(-6) M) inhibited plateau phase of slow waves and the associated increases in [Ca2+]cyt and muscle tension. BAY K 8644 (10(-7) M) augmented the plateau potential and increased [Ca2+]cyt and muscle tension. These results suggest that dihydropyridine-sensitive Ca2+ currents participate in the plateau potential. Cholinergic stimulation modulates [Ca2+]cyt and therefore force by regulating the amount of Ca2+ entering cells through these channels.

Background & Aims: The relative movements of longitudinal muscle (LM) and circular muscle (CM) and the role that nerves play in coordinating their activities has been a subject of controversy. We used fluorescent video imaging techniques to study the origin and propagation of excitability simultaneously in LM and CM of the small intestine. Methods: Opened segments of guinea pig ileum were loaded with the Ca 21 indicator fluo-3. Mucosal reflexes were elicited by lightly depressing the mucosa with a sponge. Results: Spontaneous Ca 21 waves occurred frequently in LM (1.2 s 21 ) and less frequently in CM (3.2 min 21 ). They originated from discrete pacing sites and propagated at rates 8‐9 times faster parallel (LM, 87 mm/s; CM, 77 mm/s) compared with transverse to the long axis of muscle fibers. The presence of Ca 21 waves in one muscle layer did not affect the origin, rate of conduction, or range of propagation in the other layer. The extent of propagation was limited by collisions with neighboring waves or recently excited regions. Simultaneous excitation of both muscle layers could be elicited by mucosal stimulation of either ascending or descending reflex pathways. Neural excitation resulted in an increase in the frequency of Ca 21 waves and induction of new pacing sites without eliciting direct coupling between layers. Conclusions: Localized, spontaneous Ca 21 waves occur independently in both muscle layers, promoting mixing (pendular or segmental) movements, whereas activation of neural reflexes stimulates Ca 21 waves synchronously in both layers, resulting in strong peristaltic or propulsive movements.

Fluorescence imaging is a useful tool to study the sequence an dynamics of the spread of excitability in biological tissues. Gastrointestinal muscles are particularly amenable to imaging using standard video rates because the frequency of events i slow and propagation velocities are slow. Calcium-sensitive fluorescent indicators such as fluo-3 provide effective markers of excitability because optically they exhibit high quantum yields and calcium plays important biological roles including regulating intracellular signaling and muscle contraction. Video sequences of gastrointestinal tissues demonstrate the existence of multiple preferred locations to indicate excitability. The spatial and temporal resolution of microscope-based imagin system allows pacing sites to be identified within single muscle bundles. Anisotropic conduction velocities result in spatially complex patterns of excitability where the range of propagation appears to be limited by 'collisions' with neighboring excitable events or recently activated regions. Although standard video rates are generally not sufficient to monitor more rapid excitable event such as nerve action potentials, fluorescence imaging can be used to investigate excitability mechanisms in tissues such as smooth muscles where event frequencies and propagation velocities are low.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The effects of cromakalim (BRL 34915) and its optical isomer lemakalim (BRL 38227) were investigated in intact tissue and freshly dispersed circular muscle cells from canine proximal colon. Cromakalim and lemakalim hyperpolarized resting membrane potential, shortened the duration of slow waves by abolishing the plateau phase, and decreased the frequency of slow waves. Glyburide, a K channel blocker, prevented the effect of cromakalim on slow-wave activity. The mechanisms of these alterations in slow-wave activity were studied in isolated myocytes under voltage-clamp conditions. Cromakalim and lemakalim increased the magnitude of a time-independent outward K current, but cromakalim also reduced the peak outward K current. Glyburide inhibited lemakalim stimulation of the time-independent background current. Nisoldipine also reduced the peak outward current, and in the presence of nisoldipine, cromakalim did not affect the peak outward component of current. This suggested that cromakalim may block a Ca-dependent component of the outward current. Lemakalim did not affect the peak outward current. We tested whether the effects of cromakalim on outward current might be indirect due to an effect on inward Ca current. Cromakalim, but not lemakalim, was found to inhibit L-type Ca channels; however, glyburide did not alter cromakalim inhibition of inward Ca current. We conclude that the effects of cromakalim and lemakalim on membrane potential and slow waves in colonic smooth muscle appear to result primarily from stimulation of a time-independent background K conductance. The effects of these compounds on channel activity may explain the inhibitory effect of these compounds on contractile activity.

CROMAKALIM BLOCKS CA CURRENTS AND RRUUCES K CURRENTS IN CANINE COLONIC SMOOTH MUSCLR CELLS. J.M. Post, R. Stevens, K.M. Sanders and J.R. Hume Dept. of Physiology, Univ. of Nevada, Reno, NV 89557, USA. We have investigated the effects of the putative K channel opener, cromakalim (BRL 34915), on electrical slow wave activity in intact muscles and on voltage-dependent currents in isolated smooth muscle cells from the circular layer of the canine proximal colon. In intact muscles, cromakalim (10 30 FM) reduced the upstroke amplitude and decreased the amplitude and duration of the plateau phase of slow waves. These effects were accompanied by a hyperpolarization of the membrane potential. In isolated cells, studied under voltage clamp with the patch clamp technique, cromakalim (10 30 MM) reduced the magnitude of outward K currents elicited during depolarizing test potentials. In cells pretreated with the Ca channel antagonist, nisoldipme, cromakalim failed to reduce K current amplitude. In cells exposed to K channel antagonists and impermeant ions, depolarizing voltage clamp steps elicited inward Ca currents, which were inhibited by exposure to cromakalim (10 30 PM). We conclude that cromakalim, in addition to acting as a K channel agonist, possesses significant Ca channel antagonist properties. These properties resemble those of another putative K channel agonist, minoxidil sulfate (Circ. Res. 65: 1102, 1989). The Ca channel blocking effects of eromakalim may explain some of the actions of this agent on slow waves of intact muscles. (Supported by NIH grant DK 41315 and an AHA grant-inaid)