Effects of acetylcholine (ACh) and substance P on the electrical and mechanical activities of the circular muscle layer of the canine proximal colon were studied. Because this muscle layer is bordered by two different pacemaker regions, responses from segments containing either a single pacemaker region or no pacemaker region were compared with responses of the complete muscle layer. Concentration-response relationships for ACh and substance P were similar between the various segments, suggesting that receptors for these agonists are expressed throughout the layer. The dominant contractile pattern induced by ACh and substance P in each segment was a 1- to 3-cycle/min rhythm. In a like manner, these agonists also elicited an electrical pattern in which a long-duration slow wave occurred one to three times per minute between short-duration slow waves. Low concentrations of nifedipine (0.01 microM) selectively antagonized the 1- to 3-cycle/min rhythm. In circular muscles with no pacemaker region, ACh (1 microM) caused depolarization, induced oscillations in membrane potential averaging 24 +/- 5 mV in amplitude and 2.9 +/- 0.9 cycles/min in frequency, and generated rhythmic contractions at the same frequency. This "interior" circular muscle was functionally innervated by cholinergic excitatory nerves. Exposure to ACh (1 microM) did not alter the conduction of slow waves through the thickness of the circular layer. In summary, the excitatory neurotransmitters, ACh and substance P, induce a dominant electrical and contractile rhythm throughout the circular muscle layer that is different from the spontaneous rhythms produced at either the myenteric or submucosal border.
We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical activity of intact circular smooth muscle from canine colon. TPeA (10 and 20 microM) increased slow-wave duration and "locked" the membrane potential around -30 mV plateau potential after several minutes of application, suggesting that K+ channels are essential for termination of colonic slow waves. Repolarization and normal slow-wave activity resumed after 20-30 min of washout. The patch-clamp technique was used to study the block of large-conductance Ca(2+)-activated K+ channels (BK channels) by TPeA and tetraethylammonium (TEA) in excised and cell-attached patches from isolated colonic smooth muscle cells. Channel block was characterized by a voltage-dependent dissociation constant [Kd(V)] for the binding of TEA and TPeA to a blocking site located a fraction of the distance across the membrane field (delta). The extracellular TEA binding site had a Kd(0) of 0.33 mM and a delta of 0.23. The extracellular TPeA binding site had a Kd(0) of 2.2 mM but showed significantly less voltage dependence (delta = 0.02). The intracellular binding site for TEA was of low affinity [Kd(0) = 76 mM]. Intracellular TPeA was the most potent blocker of BK channel current [Kd(0) = 11.7 microM]. The voltage dependence of block by intracellular TPeA (delta = -0.21) was not significantly different from that of intracellular TEA (delta = -0.3). Internal TPeA (10 microM) also blocked a 70-pS K+ channel and a 23-pS K+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)
We investigated the effect of phencyclidine (PCP) on three native delayed rectifier K+ currents and three channels cloned from canine and human circular colonic myocytes using voltage-clamp techniques. Native delayed rectifier K+ current in canine circular colon is composed of at least three components: (i) a rapidly activating, 4-aminopyridine-sensitive component (termed IdK(f)); (ii) a slowly activating, tetraethylammonium (TEA)-sensitive component (IdK(s)); and (iii) a rapidly activating, TEA-sensitive component, which has a steady-state inactivation curve shifted towards more negative potentials (IdK(n)). PCP blocked all three components with EC50 values of 45, 27 and 59 micromol L-1, respectively. Blocking was neither use-dependent nor voltage-dependent. Delayed rectifier K+ channels cloned from canine (Kv1.2, Kv1.5) and from human (Kv2.2) colon were expressed in Xenopus oocytes. PCP blocked all three currents with similar potency. In contrast, PCP (up to 10-4 mol L-1) did not reduce the magnitude of Ca2+-dependent outward current of large conductance Ca2+-activated K+ channels (BK channels).
Charybdotoxin (ChTX) is a specific blocker of Ca2+-activated K+ channels. The voltage- and time-dependent dynamics of ChTX block were investigated using canine colonic myocytes and the whole cell patch-clamp technique with step and ramp depolarization protocols. During prolonged step depolarizations, K+ current slowly increased in the continued presence of ChTX (100 nM). The rate of increase depended on membrane potential with an e-fold change for every 60 mV. During ramp depolarizations, the effectiveness of ChTX block depended significantly on the rate of the ramp (50% at 0.01 V/s to 80% at 0.5 V/s). Results are consistent with a mechanism in which ChTX slowly “unbinds” in a voltage-dependent manner. A simple kinetic model was developed in which ChTX binds to both open and closed states. Slow unbinding is consistent with ChTX having little effect on electrical slow waves recorded from circular muscle while causing depolarization and contraction of longitudinal muscle, which displays more rapid “spikes.” Resting membrane potential and membrane potential dynamics are important determinants of ChTX action.
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