Properties of pacemaker potentials recorded from myenteric interstitial cells of Cajal distributed in the mouse small intestine

Abstract

Recording of electrical responses from isolated small intestine of mice using conventional microelectrodes revealed two types of potential, a pacemaker potential and a slow wave, both with rapid rising primary components and following plateau components. The rate of rise and peak amplitude were greater for pacemaker potentials than for slow waves, and the plateau component was smaller in slow waves than in pacemaker potentials. Both potentials oscillated at a similar frequency (20-30 min-1). Unitary potentials often discharged during the interval between pacemaker potentials. Infusion of Lucifer Yellow allowed visualization of the recorded cells; pacemaker potentials were recorded from myenteric interstitial cells of Cajal (ICC-MY) while slow waves were recorded from circular smooth muscle cells. Pacemaker potentials were characterized as follows: the primary component was inhibited by Ni2+, Ca2+-free solution or depolarization with high-K+ solution, the plateau component was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of Ca2+-activated Cl- channels, low [Cl-]o solution or Ca2+-free solution, and the generation of potentials was abolished by co-application of Ni2+and DIDS or by chelating intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM). These results indicate that in the mouse small intestine ICC-MY generate pacemaker potentials with two components in situ; the primary and plateau components may be generated by activation of voltage-dependent Ca2+-permeable channels and Ca2+-activated Cl- channels, respectively. Slow waves are generated in circular smooth muscles via electrotonic spread of pacemaker potentials. These properties of intestinal pacemaker potentials are considered essentially similar to those of gastric pacemaker potentials.

Figure 1. Slow waves and pacemaker potentials recorded from mouse small intestine

A, a train of slow waves. B, a train of pacemaker potentials. C, a noisy pattern of pacemaker potentials. D, superimposed slow wave (a) and pacemaker potential (b) recorded at high speed (recorded from the same preparation). The resting membrane potentials were: A−77 mV, B−76 mV, C−66 mV, D−76 mV. A–D were recorded from different tissues.

Figure 3. Morphological properties of pacemaker potential generating cells after infusion of Lucifer Yellow

Aa, pacemaker potentials recorded from a cell shown in Ab. The resting membrane potential was −70 mV. The fluorescence of the injected Lucifer Yellow was viewed with a confocal microscope (Ab). Ac is an enlarged view of Ab, in which the impaled cell is indicated by the arrow. B is a confocal image of another preparation loaded with Lucifer Yellow. The calibration bars on Ab, Ac and B represent 100 μm, 20 μm and 20 μm, respectively.

Figure 2. Morphological properties of slow wave generating cells after infusion of Lucifer Yellow

Aa, slow waves recorded from a cell shown in Ab. The resting membrane potential was −65 mV. The preparation was viewed with a confocal microscope (Ab). Ac is an enlarged view of part of Ab. B is a confocal image of another preparation loaded with Lucifer Yellow. The calibration bars on Ab, Ac and B represent 200 μm, 50 μm and 100 μm, respectively.

Figure 4. Effects of NiCl₂ on pacemaker potentials recorded from mouse small intestine

Pacemaker potentials were recorded before (A) and during application of NiCl₂ (B, 10 μm; C, 30 μm; D, 60 μm). E, high speed traces of pacemaker potentials recorded in the absence (a) and presence of 60 μm NiCl₂ (b). All traces were recorded from the same cell with a resting membrane potential of −75 mV.

Figure 5. Effects of DIDS and low [Cl⁻]_o solution on pacemaker potentials recorded from mouse small intestine

Pacemaker potentials were recorded before (Aa) and during application of 2 mm DIDS (Ab). B, high speed traces of pacemaker potentials recorded in the absence (a) and presence of 2 mm DIDS (b). Pacemaker potentials were recorded before (Ca) and during application of low [Cl⁻]_o solution (Cb). D, high speed traces of pacemaker potentials recorded in the absence (a) and presence of low [Cl⁻]_o solution (b). The resting membrane potentials were: A−65 mV, C−70 mV. A and C were recorded from different tissues.

Figure 6. Effects of combined application of NiCl₂, DIDS and low [Cl⁻]_o solution on pacemaker potentials recorded from mouse small intestine

A, DIDS (2 mm) was applied to preparation (indicated by the horizontal bar) in the presence of 60 μm NiCl₂. B, NiCl₂ (60 μm) was applied to preparation (indicated by the horizontal bar) in the presence of 2 mm DIDS. C, NiCl₂ (60 μm) was applied to preparation (indicated by the horizontal bar) in the presence of low [Cl⁻]_o solution. The resting membrane potentials were: A−69 mV, B−72 mV, C−69 mV. All traces were recorded from different tissues.

Figure 7. Properties of two components of slow waves recorded from circular smooth muscle cells of mouse small intestine and the relationship between transient repolarization and dV/dt_max of slow waves

A, high speed traces of slow waves recorded in the absence (a) and presence of 60 μm NiCl₂ (b). B, high speed traces of slow waves recorded in the absence (a) and presence of 2 mm DIDS (b). C, high speed traces of slow waves recorded in the absence (a) and presence of low [Cl⁻]_o solution (b). D, large (a) and small (b) transient repolarizations after depolarization of primary component of slow waves. E, the relationship between dV/dt_max of slow waves (abscissa) and the amplitude of repolarization (ordinate). The regression line is given by Y = 0.08 + 28.5X (Y, amplitude of repolarization; X, dV/dt_max; r = 0.75, n = 128 from 8 tissues, P < 0.0001). The resting membrane potentials were: A−71 mV, B−70 mV, C−65 mV, D−72 mV. All traces were recorded from different tissues.

Figure 8. Effects of [Ca²⁺]_o-free solution on pacemaker potentials recorded from mouse small intestine

A, pacemaker potentials were recorded before (a) and during application of [Ca²⁺]_o-free solution (b). B, high speed traces of pacemaker potentials recorded in the absence (a) and presence of [Ca²⁺]_o-free solution for 22 min (b). C, [Ca²⁺]_o-free solution was applied to the preparation (indicated by the horizontal bar) in the presence of 2 mm DIDS. The resting membrane potentials were: A−70 mV, C−72 mV. A and C were recorded from different tissues.

Figure 9. Effects of BAPTA-AM on pacemaker potentials recorded from mouse small intestine

A, BAPTA-AM (50 μm) was applied (indicated by the horizontal bar). B, responses were recorded before (a) and during the application of 50 μm BAPTA-AM at 6 min (b), 12 min (c) and 37 min (d). The resting membrane potentials were: A−64 mV, B−69 mV. A and B were recorded from different tissues.

Figure 10. Effects of high K⁺ solution on pacemaker potentials recorded from mouse small intestine

Pacemaker potentials were recorded before (A) and during application of high K⁺ solution (B, 10.6 mm[K⁺]_o; C, 15.3 mm[K⁺]_o; D, 20.0 mm[K⁺]_o). All responses were recorded from the same cell with a resting membrane potential of −67 mV.

Figure 11. Effects of forskolin and SIN1 on pacemaker potentials and slow waves recorded from mouse small intestine

A and B, pacemaker potentials recorded during application of 5 μm forskolin (A) or 100 μm SIN1 (B) (applied as indicated by the horizontal bars). C and D, slow waves recorded during application of 5 μm forskolin (C) or 100 μm SIN1 (D) (applied as indicated by the horizontal bars). The resting membrane potentials were: A−73 mV, B−69 mV, C−63 mV, D−64 mV. All traces were recorded from different tissues.