Morphological and physiological evidence for interstitial cell of Cajal-like cells in the guinea pig gallbladder

Abstract

Gallbladder smooth muscle (GBSM) exhibits spontaneous rhythmic electrical activity, but the origin and propagation of this activity are not understood. We used morphological and physiological approaches to determine whether interstitial cells of Cajal (ICC) are present in the guinea pig extrahepatic biliary tree. Light microscopic studies involving Kit tyrosine kinase immunohistochemistry and laser confocal imaging of Ca(2+) transients revealed ICC-like cells in the gallbladder. One type of ICC-like cell had elongated cell bodies with one or two primary processes and was observed mainly along GBSM bundles and nerve fibres. The other type comprised multipolar cells that were located at the origin and intersection of muscle bundles. Electron microscopy revealed ICC-like cells that were rich in mitochondria, caveolae and smooth endoplasmic reticulum and formed close appositions between themselves and with GBSM cells. Rhythmic Ca(2+) flashes, which represent Ca(2+) influx during action potentials, were synchronized in any given GBSM bundle and associated ICC-like cells. Gap junction uncouplers (1-octanol, carbenoxolone, 18beta-glycyrrhetinic acid and connexin mimetic peptide) eliminated or greatly reduced Ca(2+) flashes in GBSM, but they persisted in ICC-like cells, whereas the Kit tyrosine kinase inhibitor, imanitib mesylate, eliminated or reduced action potentials and Ca(2+) flashes in both cell types, as well as associated tissue contractions. This study provides morphological and physiological evidence for the existence of ICC-like cells in the gallbladder and presents data supporting electrical coupling between ICC-like and GBSM cells. The results support a role for ICC-like cells in the generation and propagation of spontaneous rhythmicity, and hence, the excitability of gallbladder.

Figure 1. Distribution of Kit-positive cells in the duodenum and extrahepathic biliary tree

Upper left, schematic drawing of the biliary tree that illustrates the regions where photomicrographs were obtained. A and B, in the duodenum (A) and the sphincter of Oddi (B), elongated (white arrows) and multipolar (black arrows) ICC cells were observed in the muscular layers (longitudinal and circular) and in the myenteric plexus. C, the number of Kit-positive cells decreased abruptly at the junction of the sphincter of Oddi (SO) with the common bile duct (asterisk). C–E, in the rest of the common bile duct (C), hepatic duct (D) and cystic duct (E) the majority of Kit-positive cells had the morphological characteristics of mast cells (arrowheads) although a few isolated ICC-like cells were also noted (arrows). F–G, in the gallbladder neck (F) and body (G) a mixed population of ICC-like cells (arrows) and mast cells (arrowheads) were observed. Scale bar on A, 125 μm, valid for B–G.

Figure 2. Distribution of Kit-positive ICC-like cells along GBSM bundles and nerve fibres

A–B, in the gallbladder, Kit-positive ICC-like cells (arrows) were elongated and had one or two main processes that were aligned along muscle bundles. Mast cells (arrowheads) were also observed. C, the ICC-like cells were aligned along SMA-positive cells mainly at the periphery of small GBSM bundles. D, elongated Kit-positive cells were sometimes observed adjacent to PGP9.5-positive neuronal processes. Abbreviation: BV, blood vessel. Scale bar on A, 250 μm, valid for B–D.

Figure 3. Kit-positive ICC-like cells in the gallbladder

In cryostat cross-sections of full-thickness gallbladder, Kit labelling was mainly observed in the subepithelial connective tissue layer (SE; A, black arrow; and B) and in the muscularis propria (IM; A, white arrow; and C). As observed in the wholemount preparation, Kit-positive cells were elongated and had one (C) or two (B) main processes. They can be differentiated from mast cells (D–E), which have larger, rounded cell body and shorter or absent processes. Abbreviation: Epi: epithelium. Scale bars: A, 50 μm; B–E, 25 μm.

Figure 4. Ultrastrutural features of ICC-like cells in the gallbladder

A, transmission electron microscopy of cross-sections of GBSM show electron dense ICC-like cells (ICC) within a GBSM bundle of the muscularis propria. At higher magnifications, wholemount gallbladder preparation revealed that ICC-like cells were rich in mitochondria (m) and caveolae (c) and formed direct contacts (curved arrows) with surrounding cells including GBSM (SM; B–C) and other ICC-like cells (ICC; D–E) and were sometimes found nearby nerve profiles (N; F) G, direct appositions (curved arrow) between GBSM cells were also observed (SM; G). Scale bars: A and E, 500 nm; B–D, 1 μm; F, 400 μm; G, 200 μm.

Figure 5. Demonstration of GBSM cells in the bundles and associated ICC-like cells revealed by laser confocal imaging of Ca²⁺ flashes with or without Ca²⁺ waves

Ai–x, GBSM muscle bundles (arrows) with associated ICC-like cells (arrowheads). Notice cytoplasmic processes (iii–x) and appositions between ICC-like and GBSM cells (ii, iv and v). v-x, ICC-like cells (arrowheads) intimately associated with GBSM in bundles at the intersections (v–viii) or at the periphery (ix–x) of the bundles. Bi–iii, traces of Ca²⁺ flashes (arrows) and Ca²⁺ waves (arrowheads) generated from GBSM (indicated by blue circles) and ICC-like (indicated by red rectangles) cells in Ai, vi and ii, respectively. In Bi, traces of Ca²⁺ flashes and Ca²⁺ waves showing that Ca²⁺ flashes in a given bundle are synchronized, while Ca²⁺ waves are asynchronous. In Bii and iii, traces of Ca²⁺ flashes and Ca²⁺ waves showing electrical coupling between GBSM and ICC-like cells by synchronized Ca²⁺ flashes. Ca²⁺ waves are not synchronized. Scale bars: 10 μm.

Figure 7. Demonstration of the differential effects of gap junction uncouplers on Ca²⁺ transients in ICC-like cellsversusGBSM cells

After 35 min exposure to connexin P 076 (A) or 1-octanol (B), Ca²⁺ transients were abolished in GBSM cells (grey traces) but persisted in ICC-like cells (black traces). The asterisks in A indicate Ca²⁺ waves; all other Ca²⁺ transients in this figure are Ca²⁺ flashes.

Figure 6. Demonstration of the effects of the gap junction uncoupler, 1-octanol, on spontaneous action potentials and Ca²⁺ transients in GBSM cells

Within minutes of initial exposure to 1-octanol, the frequencies of action potentials (A) as well of Ca²⁺ flashes and waves (B) were dramatically reduced in GBSM cells, and Ca²⁺ flashes were abolished in the cells represented in B. In B, data from 3 GBSM cells are shown and filled circles indicate synchronized Ca²⁺ flashes.

Figure 8. Demonstration of the effects of imatinib on action potentials, Ca²⁺ flashes and Ca²⁺ waves

Traces showing that imatinib (10 μm) disrupts the discharge and rhythmic pattern and decreases the frequency of action potentials(A) and Ca²⁺ flashes (B) in intact guinea pig gallbladder muscularis. The action of imatinib on the frequency of action potentials (C 25 min, P < 0.0007), Ca²⁺ flashes and Ca²⁺ waves (D) in GBSM cells is concentration and time dependent.