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|Title:||Interaction of Two Sets of Pacemakers in Canine Ileum: Neuromodulation, Ca^2+ - Dependence, and Electrical Coupling|
|Department:||Electrical and Computer Engineering|
|Abstract:||We investigated the origins, neural modulation, ionic mechanisms, and electrical coupling properties of the pacemaker systems in the canine ileum by simultaneously recording the intracellular electrical activity and accompanying mechanical activity in cross-sectioned slabs of the muscularis extema or in the isolated circular muscle. In the whole thickness preparation, intracellular recordings were taken from the circular muscle near the myenteric plexus (MyP), deep muscular plexus (DMP), and intermediate areas between the MyP and DMP and in the isolated circular muscle preparation from similar areas except near the myenteric plexus. One type of slow wave, sigmoidal or triangular in shape, was recorded from impalements near the DMP region in the whole-thickness preparation. Another type observed from the MyP region oscillated at nearly the same frequency (9-10 cycles/min) and was characterized by a fast upstroke and a square shape. A mixture of these two patterns was recorded in intermediate areas (the outer circular muscle or OCM) while triangular slow waves were present near the the submucosal plexus (SMP) inner circular muscle. Neither type of slow waves was affected by atropine, guanethidine, propranolol, and phentolamine (all 1 μM). Under these conditions of inhibition of NANC (non-adrenergic, non-cholinergic) nerves, electrical field stimulation (EFS) produced a fast, monophasic inhibitory junction potential (IJP) followed by a triggered slow wave (TSW) which could be premature or delayed and whose amplitude was maximum near the MyP region and decayed progressively in the other areas (minimum in SMP region). The K+ channel blocker, apamin at 10⁻⁶ M, did not affect resting membrane potentials or spontaneous slow waves but inhibited the amplitude of the IJP up to 70% and slightly but significantly enhanced (30%) the amplitude of the TSW. Long duration, single pulses (50-100 msec square waves, 10-20 V) elicited TSWs without IJPs. Both the slow waves and TSWs were associated with contractions of circular muscle which were significantly enhanced by apamin but not by blockers of adrenergic and cholinergic nerves. When the IJPs recorded near the MyP or DMP were abolished by tetrodotoxin (TTX, 1 μM) or by the NO synthase (NOS) inhibitor, N^ω nitro L-arginine (L-NNA, 50 μM), the occurrence of the TSW in response to EFS was advanced in time and increased in amplitude. The effects of L-NNA were reversed by L-but not D-arginine (both 1 mM). L-arginine significantly prolonged the durations of IJPs from the MyP and DMP regions. In contrast, the N-type Ca²⁺ channel blocker ω-conotoxin GVIA (ω-CTX, 1-3 x10⁻⁷M) abolished the IJP but delayed the induction of the TSW. Subsequent addition of either TTX or L-NNA advanced the onset of the TSW. The TSWs elicited by 50-100 msec single pulses were resistant to TTX, ω-CTX, or L-NNA. All treatments which abolished the IJP significantly increased contractions of circular muscle associated with spontaneous slow waves and TSWs. In the isolated circular muscle preparation (with the DMP intact) triangular slow waves were recorded near the DMP or close to the MyP border. The frequency and amplitude of the slow waves recorded near the DMP were significantly smaller than those recorded in similar areas in the full thickness preparation. EFS of this preparation evoked IJPs of 18-20 mV in amplitude. The IJPs were biphasic, lasted 5s and showed a fast and a slow component. No TSW occurred after the fast component of the IJPs; slow repolarization was observed instead. Long duration single pulses did not induce TSWs. In this preparation, the NOS inhibitor, N^ω nitro L-arginine methyl ester (L-NAME, 3x10⁻⁴ M), abolished the IJPs and regularized the slow waves, but TSWs could not be evoked. Superfusion of inhibitory neuromediators had different effects on pacemaking activity. SIN-1, a donor of NO, hyperpolarized the membrane, significantly increased slow wave frequency but reduced its amplitude, and abolished contractions. VIP (less effective) and PACAP (more effective) reduced slow wave frequency and amplitude without changing resting membrane potentials. P ACAP, but not VIP, increased circular muscle tone at 10⁻⁶ M. Nifedipine (10⁻⁷ and 3 X 10⁻⁷ M), an L-type calcium channel blocker, did not modify the shape of slow waves in any area of the full thickness preparation. It also did not reduce the amplitude of the IJP or TSW. Ni²⁺ at 200 μM, a Ca²⁺ channel blocker, inhibited slow wave frequency and amplitude and contractions. In Ca²⁺ -free Krebs (0.1 mM EGTA) for 10-15 min, the amplitude and frequency of the slow waves were gradually reduced. The TSW in response to 100 msec single pulses was still recorded near the MyP but never near the DMP region. The inhibitory effect of Ca²⁺ -free solution on slow wave amplitude was more rapid in onset near the DMP region. The intracellular Ca²⁺ store pump inhibitor, cyclopiazonic acid (10-30 μM), enhanced slow wave frequency and contractions. This differential sensitivity to removal of Ca²⁺ may be related to the morphological and functional observations which suggested that different electrical coupling properties between the pacemaker networks existed. The MyP pacemakers were less electrically well-coupled by visible gap junctions (low resistive cell-to-cell contacts) to outer circular muscle and hence showed greater susceptibility to 1 mM octanol (a gap junction blocker). The DMP pacemakers made numerous gap junction contacts to circular muscle, and slow waves paced from this region were less susceptible to 1 mM octanol. We conclude that 1) the pacemaker system of the canine ileum consists of two types of pacemakers that correspond to the presence of two networks of pacemaker cells found in the MyP and the DMP. The MyP network appeared to dominate pacemaking activity. 2) The slow waves and the TSW originated independently of neural activity but were delayed by IJPs. The MyP and the DMP provide two independent inhibitory neural inputs, where NO is released to mediate IJPs and relaxation and influence the delay in the occurrence of the TSW. 3) The TSW originates exclusively from the MyP region from which it spreads passively to other areas. It can reset the timing of slow waves in both pacemaker networks. 4) Ca²⁺ entry through non L-or N-type Ca²⁺ channels initiates slow waves. Intracellular Ca²⁺ stores modulate slow waves. 5) Different nature of electrical coupling of the MyP and DMP pacemakers to circular muscle may explain the differential sensitivity of slow waves to Ca²⁺ removal and gap junction blockade.|
|Appears in Collections:||Digitized Open Access Dissertations and Theses|
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