PATHWAYS THROUGH THE INTERCELLULAR CLEFTS OF FROG MESENTERIC CAPILLARIES

1. The three-dimensional ultrastructure of endothelial intercellular c lefts of frog mesenteric capillaries of known hydraulic permeability ( L(p)) has been investigated in the absence and presence of lanthanum i ons as tracers of extracellular solute. 2. Experiments were carried ou t on the exposed mesenteries of pithed frogs and L(p) of a chosen micr ovessel perfused with a Ringer solution containing serum albumin (10-4 0 mg ml-1) was determined. In some experiments the mesentery was fixed in situ with 2.5% glutaraldehyde immediately after L(p) had been meas ured. In other experiments, measurement of L(p) was followed by brief microperfusion (10-20 s) with a second Ringer solution containing 1% l anthanum nitrate as a tracer before in situ fixation of the tissue. Th e tissue was prepared for electron microscopy using standard technique s. The perfused capillary was identified in the block and serial trans verse sections were cut along its length over regions where L(p) had b een measured. 3. ln six capillaries where the tissues were fixed immed iately after measurement of L(p), L(p) had a mean value (+/- S.E.M.) o f 4 (+/- 0.5) x 10(-7) cm s-1 (cmH2O)-1. Serial (30-40 nm) sections of these vessels revealed that a single short narrow region of the inter cellular clefts ran almost continuously from section to section. Addit ional tight regions were regularly seen, but they usually extended for relatively few sections. In 13.36 mum of reconstructed cleft, there w ere three interruptions of the tight region of 0.14, 0.14 and 0.17 mum respectively. In the region of these discontinuities, the wide region was uninterrupted from luminal to abluminal surface. 4. Examination o f the tight junction on a tilting stage revealed that the outer leafle ts of the adjacent cells were not fused, but separated by a gap of mea n width (+/- S.E.M.) 2.3 (+/- 0.1) nm. 5. In four capillaries perfused with lanthanum nitrate before fixation, mean L(p) (+/- S.E.M.) was 6. 5 (+/- 0.02) x 10(-7) cm s-1 (cmH2O)-1. Segments of intercellular clef ts, totalling 23.56 mum in length, were reconstructed from serial sect ions and throughout these, electron-dense deposits of lanthanum were o bserved to till the luminal parts of the intercellular clefts tip to t he tight region. Lanthanum deposits filled the entire cleft to the abl uminal surface at eleven sites, which accounted for a length of 2.52 m um out of the 23.56 mum. Only five of these regions were delimited wit hin a continuous series of sections and their mean length (+/- S.E.M.) was 0.16 (+/-0.063) mum. 6. Although there appears to be a gap betwee n the outer leaflets of the adjacent cells through the tight junction, this does not appear permeable to lanthanum. A mathematical model of flow through discontinuities of the tight regions suggests that this a lone can account for values of L(p) greater than those observed in the present study. It is noted that additional structures are necessary t o account for the sieving of macromolecules and these could take the f orm of a fibre matrix in the wide region of the cleft or at its entran ce.