Evidence supporting presence of two pacemakers in rat colon.

Intracellular microelectrodes and organ bath techniques were used to study spontaneous cyclic electrical and mechanical activity in the rat colon. Electron microscopy and immunohistochemical studies showed two major populations of interstitial cells of Cajal (ICC): one associated with Auerbach's plexus (ICC-AP) and one with the submuscular plexus (ICC-SMP). The ICC-SMP network partly adhered to the submucosa when removed and was generally strongly damaged after separation of musculature and submucosa. Similarly, longitudinal muscle removal severely damaged AP. Two electrical and mechanical activity patterns were recorded: pattern A, low-frequency (0.5--1.5 cycles/min), high-amplitude oscillations; and pattern B, high-frequency (13--15 cycles/min), low-amplitude oscillations. Pattern A was recorded in preparations with intact AP but absent in those without intact AP. Pattern B was recorded in preparations with intact SMP but was absent in those lacking SMP. With full-thickness strips, the superimposed patterns A and B were recorded in circular muscle. When longitudinal muscle mechanical activity was recorded, only pattern A was present. We conclude that two pacemakers regulate rat colonic cyclic activity: the ICC-SMP network (responsible for cyclic slow waves and small-amplitude contractions) and the ICC-AP network (which may drive the cyclic depolarizations responsible for high-amplitude contractions). This is the first report showing consistent slow wave activity in the rodent colon.

Electrical and mechanical effects of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide in the rat colon involve different mechanisms.

This work aimed to study the effects of pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) on the mechanical and electrical activity of the circular muscle of the rat colon and the mechanisms involved in such effects. Spontaneous mechanical activity was studied in vitro in an organ bath and the membrane potential was recorded using the microelectrode technique. Both VIP and PACAP (0.1 microM) caused an immediate, sustained and tetrodotoxin (1 microM)-resistant inhibition of the cyclic spontaneous mechanical activity and hyperpolarization. The small-conductance Ca(2+)-activated K(+) channel blocker, apamin (1 microM), did not change the VIP- and PACAP-induced relaxation but reduced the hyperpolarization induced by PACAP whereas it did not change that induced by VIP. In contrast, the purinoceptor antagonist, suramin (100 microM), blocked the hyperpolarization caused by PACAP and VIP but failed to change their mechanical inhibitory effects. Moreover, the putative PACAP and VIP receptor antagonists, PACAP-(6-38) and VIP-(10-28), respectively, both 3 microM, failed to change the effects of either peptide and modified neither the inhibitory junction potential nor the relaxation induced by electrical-field stimulation. Thus, these results suggest that the mechanisms mediating relaxation are not strictly coupled to the mechanisms mediating hyperpolarization. This could be due to activation of two distinct mechanisms of action after agonist receptor interaction.

Neural modulation of the cyclic electrical and mechanical activity in the rat colonic circular muscle: putative role of ATP and NO.

1. The rat colonic circular muscle displays cyclic episodes of myenteric potential oscillations (MPOs), each of them associated with a spontaneous contraction. Nifedipine 1 microM abolished both MPOs and their associated contractions. TTX (1 microM) increased the amplitude and frequency of spontaneous contractions. 2. Electrical field stimulation (EFS) induced a non-adrenergic non-cholinergic (NANC) inhibitory junction potential (IJP), with two phases: an initial fast hyperpolarization (characterized by IJP amplitude) and a sustained hyperpolarization (characterized by IJP duration). 3. Sodium nitroprusside (10 microM) hyperpolarized and abolished spontaneous contractions even in presence of TTX or 1 microM apamin. ATP (100 microM) also hyperpolarized and abolished spontaneous contractions but its effects were decreased by TTX and abolished by apamin. 4. Suramin (100 microM) or apamin reduced the amplitude of the IJPs, but did not affect their duration. Incubation with L-NOARG (1 mM) reduced the duration but not the amplitude of the IJPs. In presence of L-NOARG plus suramin or L-NOARG plus apamin, both duration and amplitude of the IJPs were reduced but a residual IJP could still be recorded. 5. We conclude that the mechanical and electrical cyclic activity of the rat colonic circular muscle is modulated but not originated by the enteric nervous system and involves L-type calcium channel activity. EFS induces release of NANC inhibitory neurotransmitters which hyperpolarize and relax smooth muscle cells. Both ATP and NO are involved in IJP generation: ATP is responsible for the first phase of the IJPs involving activation of apamin-sensitive potassium channels, whereas NO initiates the second phase which is independent of the activation of such channels.

Evidence for presence of ATP-sensitive K+ channels in rat colonic smooth muscle cells.

Coexpression of sulfonylurea receptor (SUR) and inward-rectifying K+ channel (Kir6.1 or 6.2) subunit yields ATP-sensitive K+ (K(ATP)) channels. Three subtypes of SUR have been cloned: pancreatic (SUR1), cardiac (SUR2A), and vascular smooth muscle (SUR2B). The distinct responses to K+ channel openers (KCOs) produced in different tissues may depend on the SUR isoform of K(ATP) channel. Therefore, we investigated the effects of pinacidil and diazoxide, two KCOs, on K(ATP) currents in intestinal smooth muscle cells of the rat colon (circular layer) using whole-cell voltage clamp. Pinacidil stimulated a time-independent K+ current evoked by various test potentials from a holding potential of -70 mV. The reversal potential of the stimulated current was about -75 mV, which is close to the equilibrium potential for K+ (E(K)). Both pinacidil and diazoxide dose-dependently stimulated K+ currents (evoked by ramp pulses), with EC50 values of 1.3 and 34.2 microM, respectively. The stimulated current was completely reversed by glybenclamide (3 microM). Since the EC50 values are close to those reported for vascular smooth muscle (VSM) cells, the SUR subtype may be similar to that in VSM cells, and could form the functional K(ATP) channel in rat colonic smooth muscle cells.