Spontaneous electrical pacemaker activity occurs in tunica muscularis of the gastrointestinal tract and drives phasic contractions. Interstitial cells of Cajal (ICC) are the pacemaker cells that generate and propagate electrical slow waves. We used Ca2+ imaging to visualize spontaneous rhythmicity in ICC in the myenteric region (ICC-MY) of the murine small intestine. ICC-MY, verified by colabeling with Kit antibody, displayed regular Ca2+ transients that occurred after electrical slow waves. ICC-MY formed networks, and Ca2+ transient wave fronts propagated through the ICC-MY networks at ∼2 mm/s and activated attached longitudinal muscle fibers. Nicardipine blocked Ca2+ transients in LM but had no visible effect on the transients in ICC-MY. β-Glycyrrhetinic acid reduced the coherence of propagation, causing single cells to pace independently. Thus, virtually all ICC-MYs are spontaneously active, but normal activity is organized into propagating wave fronts. Inhibitors of dihydropyridine-resistant Ca2+ entry (Ni2+ and mibefradil) and elevated external K+ reduced the coherence and velocity of propagation, eventually blocking all activity. The mitochondrial uncouplers, FCCP, and antimycin and the inositol 1,4,5-trisphosphate receptor-inhibitory drug, 2-aminoethoxydiphenyl borate, abolished rhythmic Ca2+ transients in ICC-MY. These data show that global Ca2+ transients in ICC-MYs are a reporter of electrical slow waves in gastrointestinal muscles. Imaging of ICC networks provides a unique multicellular view of pacemaker activity. The activity of ICC-MY is driven by intracellular Ca2+ handling mechanisms and entrained by voltage-dependent Ca2+ entry and coupling of cells via gap junctions.

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Background & Aims In human and canine colon, both slow (slow waves, 2–8/min) and fast (myenteric potential oscillations; MPOs, 16–20/min) electrical rhythms in the smooth muscle originate at the submucosal and myenteric borders, respectively. We used Ca2+ imaging to investigate whether ICC at these borders generated distinct rhythms. Methods Segments of canine colon were pinned submucosal or myenteric surface uppermost, or cut in cross-section. Tissues were loaded with a Ca2+ indicator (fluo-4) and activity was monitored at 36.5±0.5°C using a CCD camera. Results Rhythmic, biphasic Ca2+ transients (5–8/min), similar in waveform to electrical slow waves, propagated without decrement as a wave front through the ICC-SM network (2–5mm/s), decaying exponentially through the thickness of the CM. In contrast, rhythmic intracellular Ca2+ waves (~16/min) and spontaneous reductions in Ca2+ were observed in ICC-MY. Normally, intracellular Ca2+ waves were unsynchronized between adjacent ICC-MY, although excitatory nerve activity synchronized activity. In addition, spontaneous reductions in Ca2+ were observed that inhibited Ca2+ waves. L-NA (100µM; NO antagonist) blocked the reductions in Ca2+ and increased the frequency (~19/min) of intracellular Ca2+ waves within ICC-MY. Conclusions ICC-SM form a tightly coupled network that is able to generate and propagate slow waves. In contrast, Ca2+ transients in ICC-MY, which are normally not synchronized, have a similar duration and frequency as MPOs. Like MPOs, their activity is inhibited by nitrergic nerves and synchronized by excitatory nerves.