The ultrastructure of the region shown to be essential for pacemaking activity of the circular muscle of the canine colon was studied. This region, at the inner border of the circular muscle, consists of a network of several layers of interstitial cells of Cajal type III. These are interconnected to one another and to the adjacent circular muscle cells by numerous gap junctions. Elsewhere in circular muscle, gap junctions are rare and small. In addition, interstitial cells are in close (often less than 20 nm) contact with nerve varicosities containing large granular vesicles or sometimes small granular vesicles. The morphology of interstitial cells resembles that of others of type III. It is suggested that this arrangement of interstitial cells, circular smooth muscles, and nerves allows for a tightly coupled network of membrane oscillators to be subject to neural modulation.

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To explain that bronchial smooth muscle undergoes sustained agonist-induced contractions in a Ca(2+)-free medium, we hypothesized that caveolae in the plasma membrane (PM) contain protected Ca(2+). We isolated caveolae from canine tracheal smooth muscle by detergent treatment of PM-derived microsomes. Detergent-resistant membranes were enriched in caveolin-1, a specific marker for caveolae as well as for L-type Ca(2+) channels and Ca(2+) binding proteins (calsequestrin and calreticulin) as determined by Western blotting. Also, the PM Ca(2+) pump was present but not connexin 43 (a noncaveolae PM protein), the sarcoplasmic reticulum (SR) Ca(2+) pump, or the type 1 inositol 1,4, 5-trisphosphate receptor, supporting the idea that SR-derived membranes were not present. Antibodies to caveolin coimmunoprecipitated caveolin with calsequestrin or calreticulin. Thus some of the cellular calsequestrin and calreticulin associated with caveolin on the cytoplasmic face of each caveola. Immunohistochemistry of tracheal smooth muscle crysosections confirmed the localization of caveolin and the PM Ca(2+) pump to the cell periphery, whereas the SR Ca(2+) pump was located deeper in the cell. The presence of L-type Ca(2+) channels, the PM Ca(2+) pump, and the Ca(2+) bindng proteins calsequestrin and calreticulin in caveolin-enriched membranes supports caveola involvement in airway smooth muscle Ca(2+) handling.

We examined the structural and functional basis for pacemaking by interstitial cells of Cajal (ICC) in circular smooth muscle of the canine ileum. Gap junctions were found between ICC of myenteric plexus (MyP), occasionally between MyP ICC and outer circular smooth muscle cells, between individual outer circular smooth muscle cells, between them and ICC of the deep muscular plexus (DMP), and between DMP ICC. No visible gap junctions connected MyP ICC to longitudinal muscle cells or inner circular muscle cells. Occasionally contacts occurred between the two muscle layers. No special structures were found to connect MyP and DMP ICC networks. Octanol concentration dependently reduced the amplitude and frequency of, but did not abolish, slow waves in circular muscle in isolated ileum recorded near the MyP or the DMP. Slow waves triggered from MyP ICC by a current pulse also persisted. Contractile activity was abolished, cells were depolarized, and fast inhibitory junction potentials were reduced by octanol. We conclude that ICC pacemakers of the MyP and DMP utilize gap junctional conductances for pacemaking function but may not require them. Coupling between the two ICC networks may utilize the circular muscle syncytium.

The hypothesis that gap junction (GJ) formation between myometrial cells at term improves electrical coupling was tested. We measured the spread of electrical excitation from six extracellular electrodes aligned on uterine strips in either the longitudinal (axial) or transverse (circumferential) direction. Spontaneous bursts propagated over the entire 15-mm recording distance in the axial direction at both preterm and parturition and showed some characteristics of a system of coupled relaxation oscillators. However, individual spikes within the bursts propagated further and with higher velocity at parturition than at preterm. In the circumferential direction, both bursts and individual spikes propagated further at parturition than before. Propagation in this axis at parturition appeared to require an intact circular muscle layer. Spikes evoked by electrical stimulation also propagated further and with higher velocity in both axes at parturition. Electron microscopy showed many GJs between uterine smooth muscle cells during parturition, but few and sometimes no GJs at preterm. Thus improved propagation was associated with increased GJ contact between myometrial cells, consistent with the hypothesis that gap junction formation at term improves electrical coupling.