Identification of interstitial cells of Cajal in the rabbit portal vein

Two layers of interstitial cells (ICs) of Cajal were detected by c-kit and methylene blue staining in the media of the rabbit portal vein in subendothelial intramuscular and deeper intramuscular positions, displaced radially from each other by about 40-70 microm. Two morphologically distinct types of ICs were found among enzymatically dispersed cells from this vessel: small multipolar cells with stellate-shaped bodies not exceeding 20 microm, and spindle-shaped cells from 40 to 300 microm in length with numerous branching processes. Relaxed smooth muscle cells (SMCs) had a more constant length (90-150 microm). The cell membrane capacitance was 46.5+/-2.2 pF in SMCs, 39.7+/-2.4 pF in spindle-shaped ICs and 27.8+/-0.7 pF in multipolar ICs. Although darker under phase contrast, after loading with fluo-4 AM, single isolated ICs of both types usually had brighter fluorescence than SMCs and displayed various spontaneous calcium events, including Ca(2+) sparks and Ca(2+) waves. Ca(2+) waves were usually followed by contraction of SMCs but no change in shape of ICs. In some ICs spontaneous [Ca(2+)](i) transients (lasting about 2s) which propagated towards the end of the processes were observed. Physical contacts between the processes of ICs and the body of one or more SMCs survived the isolation procedure. Application of noradrenaline (1-10 microM), caffeine (1-10 mM) or high-K(+) solution (60mM) led to a rise of [Ca(2+)](i) in both SMCs and ICs evoking contraction of SMCs but not ICs. No differences in electrophysiological characteristics between single enzymatically isolated IC and SMC were detected; thus, the resting membrane potential estimated under current-clamp conditions was -46.5+/-2.0 mV in spindle-shaped ICs and -45.6+/-2.7 mV in SMCs. Under voltage-clamp, both ICs and SMCs revealed a well-developed voltage-gated nifedipine-sensitive L-type Ca(2+) current, a set of K(+) currents, including spontaneous transient outward currents (STOCs) but no Na(+) current. This study for the first time directly demonstrated the presence in vascular tissue of ICs. Possible roles for ICs including their involvement in spontaneous activity of the vessel were discussed.