The rabbit portal vein discharges spontaneous action potentials accompanied by contractions when the whole vein, or a portion of it, is bathed in physiological salt solution. This spontaneous activity is myogenic and seems to be due to the presence of multiple pacemakers in the wall of the portal vein (Sutter, 1990). Enzymatic dispersion of the rabbit portal vein revealed two types of cells: more numerous smooth muscle cells (SMCs) were mixed with unusual cells morphologically resembling closely the interstitial cells of Cajal, which were shown to be specialised pacemaking cells in the gut (Sanders et al. 1999). Their presence in blood vessels has not been previously reported and we were intrigued to investigate their features, functions and location in the portal vein.

A 20 mm section of portal vein was removed from male New Zealand rabbits (2-3 kg) killed by an overdose of pentobarbitone. Experiments were performed at room temperature on segments of the opened vein after removal of fat and the adventitial layer, or on freshly isolated single cells within a few hours of enzyme and mechanical dispersion. Single cells were studied with standard whole-cell and amphotericine B perforated patch methods or imaged after loading with the fluorescent Ca²⁺-sensitive indicator fluo-4 AM. c-kit-labelled living tissue was obtained as described elsewhere (Hirst & Edwards, 2001): portal vein segments were incubated with ACK-2 rat monoclonal antibodies (from Gibco BRL or from Santa Cruz) and then with Alexa Fluor 633 conjugated anti-rat antibodies (Molecular Probes). In controls, primary antibodies were omitted from the first incubation solution. All single-cell or whole tissue imaging was performed using a laser scanning confocal microscope.

In contrast to relaxed SMCs, which have a more constant shape (90-150 mm in length with a mean of 121 ± 23 mm and about 8-10 mm in width with a mean of 8.8 ± 1.1 mm; ± S.D., n = 47), interstitial cells varied somewhat in their morphology (from 40 to 300 mm in length with a mean of 147 ± 51 mm and from 4 to 12 mm in width with a mean of 7.2 ± 2.0 mm; ± S.D., n = 98), but they always had a variable number (from several to several tens) of numerous, branching, extremely thin (often less than 1 mm wide) processes which could be up to several tens of micrometres in length. Interstitial and SMCs had no differences in electrophysiological characteristics. Under voltage clamp both cell types revealed well-developed voltage-gated nifedipine-sensitive L-type Ca²⁺ current but no Na⁺ current. After loading with fluo-4 AM SMCs, and less commonly, interstitial cells, displayed various spontaneous calcium events including Ca²⁺ sparks and Ca²⁺ waves. Ca²⁺ waves were followed by contraction of the SMCs, but not interstitial cells. The latter usually had brighter fluorescence than SMCs but appeared darker under phase contrast. Application of noradrenaline (1Ð10 mM), caffeine (1Ð10 mM) or high K⁺ solution (60 mM) evoked a rise of [Ca²⁺]_i in both cell types and a contraction of SMCs, but no change in the shape of interstitial cells was observed. Some interstitial cells showed spontaneous [Ca²⁺]_i transients (lasting about 2 s) propagating from the cell body to the end of the processes. Surviving contacts between the processes of interstitial cells and SMCs could be found after the cell isolation. Portal vein tissue showed cells stained with Methylene Blue and c-kit-positive cells in the media about 7Ð30 mm from the endothelium.

It is possible that these interstitial cells could be responsible, as in gut muscles, for driving its spontaneous activity.

This work was supported by the British Heart Foundation (RG/99001) and The Wellcome Trust (042293 and 064786).

All procedures accord with current UK legislation.