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.
The effects of nitric oxide (NO) on intracel- collected by using a video-based imaging system that has been described (23). The locations of cells of interest were specified by using a computer mouse and custom-designed software. Signals were recorded as the fluorescence within a rectangular region over each cell. Fluorescent responses were normalized by dividing by the maximum response to the bath perfusion of a solution containing 1 ,uM Bay K 8644. Thus, the ratio associated with the ordinate of each trace represents a fraction of the increase in [Ca2+]i relative to the response to 1 ,uM Bay K 8644. By continuously framegrabbing images, the computer was able to track the activity of a number of individual cells within a field of view simultaneously. NO was introduced by bolus application of saturated solution (1 mM) to the bath. Bay K 8644 was applied to individual cells by micropressure ejection (2 bars; 15 msec) using a Picospritzer apparatus (General Valve, Fairfield, NJ). Other agents were added or removed from solutions by rapidly exchanging bath medium. MATERIALS AND METHODS CellsHanks' perfusion buffer, NO stock solutions, oxyhemoglobin, and methemoglobin were prepared as described (11, Abbreviations: GI, gastrointestinal; IC, interstitial cell; SMC, smooth muscle cell; [Ca2+]j, intracellular Ca2+ concentration; L-NAME, NG-nitro-L-arginine methyl ester. 2087The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Rotating waves (rotors) of cellular activity were observed in nonconf luent cultures of embryonic chick heart cells by using a macroscopic imaging system that detected f luorescence from intracellular Ca 2؉ . Unlike previous observations of rotors or spiral waves in other systems, the rotors did not persist but exhibited a repetitive pattern of spontaneous onset and offset leading to a bursting rhythm. Similar dynamics were observed in a cellular automaton model of excitable media that incorporates spontaneous initiation of activity, and a decrease of excitability as a consequence of rapid activity (fatigue). These results provide a mechanism for bursting dynamics in normal and pathological biological processes.Since early studies demonstrated that bursting rhythms could originate from single cells (1), theoretical analyses of bursting have focused on cellular ionic mechanisms (2-5). It has been difficult to assess what contribution spatial patterns of activation within groups of participating cells have on the dynamics of a burst. This work was initiated to determine the spatial patterns of activation occuring in dispersed cultures of heart cells. Previous studies of excised slices of cardiac tissue demonstrated sustained rotors initiated by electrical stimulation and proposed that rotors might be a mechanism for abnormally rapid cardiac arrhythmias (tachycardias) (6, 7). Although tachycardias in people can be initiated by electrical stimulation, they usually start spontaneously. In most instances, the rhythms also terminate spontaneously. On occasion there is a repeated spontaneous paroxysmal initiation and termination of tachycardias (8,9). In this paper, we demonstrate repeated spontaneous initiation and termination of rotors in cultured cardiac myocytes.These phenomena are modeled by using a cellular automaton. In a cellular automaton, there is a lattice of sites with discrete states and a synchronous updating rule that is identical for all sites. Cellular automata have been used as simplified models for a large range of phenomena, including neural networks (10), cardiac arrhythmias (11), oscillating chemical reactions (12), and forest fires (13). However, none of these previous formulations display the repeated onset and offset of rotors observed here. METHODSCell Culture. Ventricular cells from 7-day embryonic chick ventricular myocytes were isolated as described (14). Cells were plated at just under confluent densities of 7.0-14.0 ϫ 10 3 cells per cm 2 within 12-mm diameter glass retaining rings on lysine-coated plastic cell culture dishes. Cells were kept in maintenance medium 818a at 5% CO 2 at 36°C for 1-2 days before experiments. Monolayers were loaded with calciumsensitive dye fluo-3 (5 M, Molecular Probes) (15) in Hanks' balanced salt solution (HBSS, 1.3 mM K) for 25-30 min, washed with fresh HBSS to remove excess dye, and then transferred to the imaging setup. Experiments were conducted at 36°Ϯ 1°C.Fluorescent Imaging. A macroscope was constructed (16) to allow low light level measurements...
Electrical slow waves recorded from circular muscle cells near the myenteric and submucosal plexuses were found to be significantly different. By measuring the latencies between the arrival of evoked events at two recording sites, slow wave conduction velocities were determined in the three dimensions of circular muscle. Slow waves propagated more rapidly in the axis parallel to the circular fibers than in the axes perpendicular to the circular fibers. The rates of slow wave propagation were also determined in axes parallel and perpendicular to fibers in myenteric and submucosal circular muscles. Slow waves conducted more slowly in the circular muscle near the submucosa than in circular muscle near the myenteric plexus. From conduction velocity measurements, a technique was developed to determine the pacemaker site of spontaneous slow waves in a muscle strip. These data demonstrate that slow waves originate from multiple discrete foci; in muscle strips cut along the long axis of the stomach, these foci are found predominantly in the orad region of the muscle strip; and slow waves originate in the outer myenteric half of the muscle.
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