Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal.

Located within the tunica muscularis of the gastrointestinal (GI) tract are networks of cells known as interstitial cells of Cajal (ICC). ICC are critical for important basic functions of GI motility such as generation and propagation of slow-wave pacemaker activity and reception of regulatory inputs from the enteric nervous system. We have developed a novel procedure to identify and isolate individual ICC from freshly dispersed cell preparations of the murine small intestine and gastric fundus and to determine differential transcriptional expression We have compared the expression profiles of pacemaker ICC isolated from the murine small intestine (IC-MY) and ICC involved in neurotransmission from the gastric fundus (IC-IM). We have also compared expression profiles between ICC and smooth muscle cells (SMC) and between freshly isolated ICC and cultured ICC. Cultured ICC express smooth muscle myosin, whereas freshly dispersed ICC do not. All cell types express muscarinic receptor types M(2) and M(3), neurokinin receptors NK(1) and NK(3), and inhibitory receptor VIP-1, whereas only cultured ICC and SMC express VIP-2. Both cultured and freshly dispersed IC-IM and IC-MY express the soluble form of stem cell factor, whereas SMC from the gastric fundus express only the membrane-bound form.

Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves.

BACKGROUND & AIMS: Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. METHODS: Glial cell line-derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. RESULTS: ICC were normally distributed throughout the gastrointestinal tracts of GDNF-/- mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. CONCLUSIONS: These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development.

Molecular diversity of K(V) alpha- and beta-subunit expression in canine gastrointestinal smooth muscles.

Voltage-activated K(+) (K(V)) channels play an important role in regulating the membrane potential in excitable cells. In gastrointestinal (GI) smooth muscles, these channels are particularly important in modulating spontaneous electrical activities. The purpose of this study was to identify the molecular components that may be responsible for the K(V) currents found in the canine GI tract. In this report, we have examined the qualitative expression of eighteen different K(V) channel genes in canine GI smooth muscle cells at the transcriptional level using RT-PCR analysis. Our results demonstrate the expression of K(V)1.4, K(V)1.5, K(V)1.6, K(V)2.2, and K(V)4.3 transcripts in all regions of the GI tract examined. Transcripts encoding K(V)1.2, K(V)beta1.1, and K(V)beta1.2 subunits were differentially expressed. K(V)1.1, K(V)1.3, K(V)2.1, K(V)3.1, K(V)3.2, K(V)3.4, K(V)4.1, K(V)4.2, and K(V)beta2.1 transcripts were not detected in any GI smooth muscle cells. We have also determined the protein expression for a subset of these K(V) channel subunits using specific antibodies by immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry demonstrated that K(V)1.2, K(V)1.4, K(V)1.5, and K(V)2.2 are expressed at the protein level in GI tissues and smooth muscle cells. K(V)2.1 was not detected in any regions of the GI tract examined. These results suggest that the wide array of electrical activity found in different regions of the canine GI tract may be due in part to the differential expression of K(V) channel subunits.

Contribution of delayed rectifier potassium currents to the electrical activity of murine colonic smooth muscle.

1. We used intracellular microelectrodes to record the membrane potential (Vm) of intact murine colonic smooth muscle. Electrical activity consisted of spike complexes separated by quiescent periods (Vm approximately -60 mV). The spike complexes consisted of about a dozen action potentials of approximately 30 mV amplitude. Tetraethylammonium (TEA, 1-10 mM) had little effect on the quiescent periods but increased the amplitude of the action potential spikes. 4-Aminopyridine (4-AP, >= 5 mM) caused continuous spiking. 2. Voltage clamp of isolated myocytes identified delayed rectifier K+ currents that activated rapidly (time to half-maximum current, 11.5 ms at 0 mV) and inactivated in two phases (tauf = 96 ms, taus = 1.5 s at 0 mV). The half-activation voltage of the permeability was -27 mV, with significant activation at -50 mV. 3. TEA (10 mM) reduced the outward current at potentials positive to 0 mV. 4-AP (5 mM) reduced the early current but increased outward current at later times (100-500 ms) consistent with block of resting channels relieved by depolarization. 4-AP inhibited outward current at potentials negative to -20 mV, potentials where TEA had no effect. 4. Qualitative PCR amplification of mRNA identified transcripts encoding delayed rectifier K+ channel subunits Kv1.6, Kv4.1, Kv4.2, Kv4.3 and the Kvbeta1.1 subunit in murine colon myocytes. mRNA encoding Kv 1.4 was not detected. 5. We find that TEA-sensitive delayed rectifier currents are important determinants of action potential amplitude but not rhythmicity. Delayed rectifier currents sensitive to 4-AP are important determinants of rhythmicity but not action potential amplitude.