Recent studies in our laboratory have demonstrated the presence of interstitial cells (ICs) in the wall of rabbit portal vein (Povstyan et al. 2003); ICs are considered to play a pacemaker role in the gastrointestinal tract and urethra (Sanders et al. 1999, Sergeant et al. 2000). Following enzymatic dispersion of portal vein fragments, isolated ICs, as well as ICs contacting other ICs or smooth muscle cells (SMCs) were obtained. We therefore investigated whether any signal transmission from IC to IC or from IC to SMC is possible through contacts which survive the isolation procedure. This was explored by combining stimulation of an IC under tight-seal voltage clamp with simultaneous imaging of [Ca2+]i in the IC and in adjacent IC(s) or SMC(s). Imaging was performed using fast x-y laser-scanning confocal microscopy of fluo-3 fluorescence.
A section of portal vein about 20 mm long, upstream from the anastomosis of its right and left branches was removed from male New Zealand rabbits (2-3 kg) immediately after they had been humanely killed by an overdose of pentobarbitone injected into the ear vein. For scanning electron microscopy, isolated cells were fixed with cacodylate buffer, post-fixed in 1 % osmium tetroxide, dehydrated and mounted on aluminum stubs. For viewing under a Zeiss EM 940 electron microscope, specimens were shadowed with a thin layer of evaporated gold that gives the secondary electron image seen on the monitor.
Contacts that survived the isolation procedure between the processes of ICs and SMCs, were observed in electron micrographs. Depolarization of an IC under voltage clamp resulted in increase in fluo-3 fluorescence in the IC without apparent latency, while a rise of [Ca2+]i in an adjacent SMC occurred also but with a delay of up to ~3 s. Thus, any evoked change in [Ca2+]i in a SMC required a long-lasting depolarization of the IC connected to it. Long-lasting spontaneous depolarizations were observed in isolated ICs under current clamp conditions. Finally, when the same protocol as was used to study IC-SMC pairs was applied to small fragments of surviving IC network, it was revealed that signal transmission from IC to IC occurred almost instantaneously.
These results suggest that the IC network may serve as a generator and/or conductor of low frequency electrical signals (such as slow wave), which act as SMC pacemakers.
Ths work was supported by Wellcome Trust grants 042293, 060659 and 064786.