Properties of spontaneously active cells distributed in the submucosal layer of mouse proximal colon

Abstract

Intracellular electrical activity was recorded from smooth muscle tissues of the mouse proximal colon, and the impaled cells were visualized by injection of neurobiotin. Slow potentials with initial fast and subsequent plateau components (plateau potentials), generated at a frequency of 14.8 min(-1), were recorded from oval-shaped cells with bipolar processes. Periodic bursts of spike potentials (4.6 min(-1)) and bursts of oscillatory potentials (4.3 min(-1)) were recorded in circular and longitudinal smooth muscle cells, respectively. Nifedipine (0.1 microM) abolished the bursts of spike and oscillatory potentials and reduced the duration of plateau potentials. The plateau potentials were abolished by 1 microM nifedipine. The plateau potentials were also abolished by cyclopiazonic acid (an inhibitor of Ca(2+) uptake into internal stores) or 2-aminoethoxydiphenyl borate (an inhibitor of inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release), and were inhibited by bis-(aminophenoxy) ethane-N,N,N,'N'-tetraacetic acid acetoxymethyl ester (a chelator of intracellular Ca(2+)). Carbonyl cyanide m-chlorophenylhydrazone (a mitochondrial protonophore) abolished plateau potentials, and its action was not mimicked by oligomycin (an inhibitor of mitochondrial ATPase). It is concluded that in mouse proximal colon, submucosal c-kit-positive bipolar cells spontaneously generate plateau potentials with rhythms different from those generated by smooth muscle cells. The plateau potentials are generated through activation of voltage-gated Ca(2+) channels, which are coupled to the release of Ca(2+) from the internal stores and the handling of Ca(2+) in mitochondria.

Figure 1. Spontaneous activity recorded from mouse proximal colon

Representative electrical activity recorded from submucosal interstitial cells (A), circular (B) and longitudinal smooth muscle cells (C). Responses recorded from tissue with mucosal layer uppermost and intact submucosal layer attached (A), tissue with circular smooth muscle layer uppermost and no submucosal layer (submucosal side uppermost, B), and tissue with longitudinal muscle uppermost, without the submucosal layer (C) are shown on different time scales: a, slow speed; b, fast speed. The resting membrane potentials in A, B and C were −50, −45 and −50 mV, respectively. Each pair of responses was recorded from the same cell.

Figure 2. Morphology of spontaneously active cells in the submucosal layer

A, neurobiotin-filled cells distributed in the submucosal layer (a and b are from different pieces of tissue). Membrane potentials were recorded from tissues of the mouse proximal colon, in the presence of 20 μm 18β-glycyrrhetinic acid for 15 min, and then neurobiotin was injected into the recorded cell by applying a depolarizing current for 5 min. B, c-kit protein-positive cells distributed in the submucosal layer (a and b are from different regions in the same preparation). The preparation was viewed with the aid of a confocal microscope (magnification × 630). Circular muscle bundles run in a vertical direction. Calibration bar / 100 μm. In Aa, the two-headed arrow indicates the length of the cell body.

Figure 3. Morphology of spontaneously active cells in the circular and longitudinal muscle layers

Membrane potentials were recorded from smooth muscle tissues of mouse proximal colon in the presence of 20 μm 18β-glycyrrhetinic acid for 15 min. Neurobiotin was then injected into the recorded cell by applying a depolarizing current for 5 min. Panels show neurobiotin-injected cells located in the circular smooth muscle bundles (A), and longitudinal smooth muscle bundles (B) of mouse proximal colon. Circular muscle bundles run in the vertical direction. Calibration bar / 100 μm. The preparations were viewed with the aid of a confocal microscope at different magnifications (Aa and Ba, × 200; Ab and Bb, × 630).

Figure 4. Effect of nifedipine on plateau potentials, spike potentials and oscillatory potentials

A, plateau potentials were recorded in the absence (Aa and Ba) and presence of nifedipine (Ab and Bb, 0.1 μm; Ac, 1 μm) from the uppermost tissue of the submucosal layer. Records in B are shown on an expanded time scale. C, bursts of spike potentials (a and b) or oscillatory potentials (c and d) were recorded from tissues in which the circular muscle or longitudinal muscle was uppermost, respectively, in the absence (a and c) and presence of nifedipine (b and d; 0.1 μm). Responses in A and B, and Cab and Ccd were obtained from different pieces of tissue. The resting membrane potentials were: A and B, −50 mV; Cab, −50 mV; Ccd, −51 mV.

Figure 5. Modulation of internal Ca²⁺ stores and plateau potentials

Plateau potentials were recorded from tissues in which the submucosal layer was uppermost, in the control solution (a) and after exposure to solution containing 1 μm cyclopiazonic acid (CPA) for 10 min (Ab), 20 μm bis-(aminophenoxy) ethane-N,N,N‘,N‘-tetraacetic acid acetoxymethyl ester (BAPTA AM) for 15 min (Bb) or 10 μm 2-aminoethoxydiphenyl borate (2-APB) for 10 min (Cb). Each pair of responses in A-C was recorded from single cells in different pieces of tissue.

Figure 6. Effects of CCCP on plateau potentials

In the uppermost layer of the submucosal layer, plateau potentials were recorded in control solution (Aa) and after 10 min exposure to a solution containing 1 μm carbonyl cyanide m-chlorophenylhydrazone (CCCP; Ab). In different pieces of tissue, plateau potentials were recorded in control solution (Ba) and after 10 min exposure to solution containing 10 μm glibenclamide (Bb) and 10 μm glibenclamide with 1 μm CCCP for 10 min (Bc). All responses in A and B were obtained from different single cells.