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Epithelium of toad urinary bladder: The assessment of tight junctions
Micron,
Voi.13, No.3, pp.375-376, Printed in Great Britain
1982.
0047-7206/82/030375-02503.00/0 Pergamon Press Ltd.
EPITHELIUM OF TOAD URINARY BLADDER:
Plumstead, Shelley M.;
THE ASSESSMENT OF TIGHT JUNCTIONS.
Rayns, D.G.;
Leader, J.P. & Macknight,A.D.C.
Otago Medical School, P.O.Box 913, Dunedin, N.Z.
Most epithelial cells are bonded by tight junctions (occluding or limiting junctions) formed by the interaction of the membranes of contiguous cells. These junctions are wide-ranging in form. They may be simple one- or two-stranded types (e.g. renal proximal convoluted tubule), three- to six-stranded intermediate types (e.g. small intestine) or seven- or more stranded extensive types such as renal collecting tubule.l These junctional strands are composed of intra-membranous protein fibrils and it is widely accepted that there are matched (paired) arrays of these fibrils within the lipid bilayers of the contributing membranes.2 In general the more numerous the strands, the tighter the junction within the paracellular pathway. However, there are exceptions, for example, cells of the avian salt gland. 3 Furthermore, the characteristics of some junctions can be varied experimentally. Toad urinary bladder is, under normal conditions, both morphologically and physiologically tight, (6 - ii junctional strands and low paracellular conductance).
By applying a 240mM hyperosmotic urea solution to the mucosal (luminal) surface of this epithelium, the paracellular conductance is increased. 4 A similar increase can be induced by applying a direct current across the epithelium. 5 We have shown that these treatments produce quantitative changes in the junctional morphology as seen by freeze-fracture electron microscopy.
A number of different approaches can be made to assess the structural changes in these junctions as seen on electron micrographs. It is possible to determine: i.
The number of approximately circumferential easy in a junction with complex geometry).
2.
The depth of the junction: the extent of the strands in the apical-basal direct ion. 6 (The average of many transects across the junctional belt.)
3.
The area of the junction per unit of junctional length. 6 formation to 2 above).
4.
The total strand length per unit of junctional length. 6 tion of density of strands in the junction).
5.
The number of strand intersections per unit area. 7 geometric complexity of the junction).
strands per junction. 1
(This is not
(This gives similar in-
(This gives an indica-
(This gives a measure of the
Each of these approaches has advantages and disadvantages. Method l, where appropriate, (e.g. distal convoluted tubule), is still the simplest and most meaningful method of describing the appearance of the junction. In more complex junctions (e.g. toad urinary bladder), method 5 gives the most useful information, improved further if related to junct-
376
S.M.
Plumstead c~ c~.
ional depth. All five methods are averaging techniques. They fail to bring out geometrical differences between the apical and basal extremities of a junction. Such differences may be minor (e.g. small intestine) or major (e.g. liver). 8 One solution is to divide the transects into three equal parts and using method 1 record the distribution of strand intersections within each zone. Symmetrical junctions show an even spread, whereas more asymmetric junctions have a definite skewed distribution.
Following the application of hyperosmotic urea or electrical stimulation (voltage clamping) noted above, we found the following structural responses in the epithelial junctions of toad urinary bladder. (Junctional depth did not change significantly).
Using method 4. Treatment Control Hyperosmotic urea Direct Current
120 ~m of strand per 10~m of junctional length 114 um of strand per 10~m of junctional length 146 ~m of strand per 10~m of junctional length
This suggests that some strand material may be generated following electrical stimulation.
Using method 5. Treatment Control Hyperosmotic urea Direct Current
2.7 strand intersections per 0.01 ~m~ of junct. 1.3 strand intersections per 0.01 ~m of junct. 1.8 strand intersections per 0.0i ~m 2 of junct.
These results suggest that as paracellular conductance is increased, the geometry of the junction becomes less complex.
Although there is the possibility of some new strand material being formed after D.C treatment, the general response of the junction to 1 hour of either specific treatment is a rearrangement of existing strand material and this is most readily detected by analysis using method 5.
References.
i. 2. 3. 4. 5. 6. 7. 8.
Claude, P. & Goodenough, D.A. 1973. J.Cell Biol. 58, 390-400. Bullivant, S. 1981. 'Epithelial Ion & Water transport.' Ed. Macknight, Raven Press, N . Y . p . 2 6 5 - 2 7 5 . Riddle, C.V. & Ernst, S.A. 1979. J. Memb. Biol. 45, 21-35. Wade, J.B. & Karnovsky, M.J. 1974. J. Cell Biol. 62, 344-350 Bindslev, N. et a 1 1 9 7 4 . Biochim. Biophys. Acta. 33__2, 286-297. Joyoe, P.R. et a 1 1 9 7 6 . Proc. Univ. Otago Med. Sch. 5_44. 47-48. Murphy, C.R. et al. 1981. Cell Biophys. ~, 57-69. Hull, B.E., Staehelin L.A. 1976. J. Cell Biol. 68. 688-704.
Acknowledgement. The authors thank the Medical Research Council of N.Z. for financial support.
Voi.13, No.3, pp.375-376, Printed in Great Britain
1982.
0047-7206/82/030375-02503.00/0 Pergamon Press Ltd.
EPITHELIUM OF TOAD URINARY BLADDER:
Plumstead, Shelley M.;
THE ASSESSMENT OF TIGHT JUNCTIONS.
Rayns, D.G.;
Leader, J.P. & Macknight,A.D.C.
Otago Medical School, P.O.Box 913, Dunedin, N.Z.
Most epithelial cells are bonded by tight junctions (occluding or limiting junctions) formed by the interaction of the membranes of contiguous cells. These junctions are wide-ranging in form. They may be simple one- or two-stranded types (e.g. renal proximal convoluted tubule), three- to six-stranded intermediate types (e.g. small intestine) or seven- or more stranded extensive types such as renal collecting tubule.l These junctional strands are composed of intra-membranous protein fibrils and it is widely accepted that there are matched (paired) arrays of these fibrils within the lipid bilayers of the contributing membranes.2 In general the more numerous the strands, the tighter the junction within the paracellular pathway. However, there are exceptions, for example, cells of the avian salt gland. 3 Furthermore, the characteristics of some junctions can be varied experimentally. Toad urinary bladder is, under normal conditions, both morphologically and physiologically tight, (6 - ii junctional strands and low paracellular conductance).
By applying a 240mM hyperosmotic urea solution to the mucosal (luminal) surface of this epithelium, the paracellular conductance is increased. 4 A similar increase can be induced by applying a direct current across the epithelium. 5 We have shown that these treatments produce quantitative changes in the junctional morphology as seen by freeze-fracture electron microscopy.
A number of different approaches can be made to assess the structural changes in these junctions as seen on electron micrographs. It is possible to determine: i.
The number of approximately circumferential easy in a junction with complex geometry).
2.
The depth of the junction: the extent of the strands in the apical-basal direct ion. 6 (The average of many transects across the junctional belt.)
3.
The area of the junction per unit of junctional length. 6 formation to 2 above).
4.
The total strand length per unit of junctional length. 6 tion of density of strands in the junction).
5.
The number of strand intersections per unit area. 7 geometric complexity of the junction).
strands per junction. 1
(This is not
(This gives similar in-
(This gives an indica-
(This gives a measure of the
Each of these approaches has advantages and disadvantages. Method l, where appropriate, (e.g. distal convoluted tubule), is still the simplest and most meaningful method of describing the appearance of the junction. In more complex junctions (e.g. toad urinary bladder), method 5 gives the most useful information, improved further if related to junct-
376
S.M.
Plumstead c~ c~.
ional depth. All five methods are averaging techniques. They fail to bring out geometrical differences between the apical and basal extremities of a junction. Such differences may be minor (e.g. small intestine) or major (e.g. liver). 8 One solution is to divide the transects into three equal parts and using method 1 record the distribution of strand intersections within each zone. Symmetrical junctions show an even spread, whereas more asymmetric junctions have a definite skewed distribution.
Following the application of hyperosmotic urea or electrical stimulation (voltage clamping) noted above, we found the following structural responses in the epithelial junctions of toad urinary bladder. (Junctional depth did not change significantly).
Using method 4. Treatment Control Hyperosmotic urea Direct Current
120 ~m of strand per 10~m of junctional length 114 um of strand per 10~m of junctional length 146 ~m of strand per 10~m of junctional length
This suggests that some strand material may be generated following electrical stimulation.
Using method 5. Treatment Control Hyperosmotic urea Direct Current
2.7 strand intersections per 0.01 ~m~ of junct. 1.3 strand intersections per 0.01 ~m of junct. 1.8 strand intersections per 0.0i ~m 2 of junct.
These results suggest that as paracellular conductance is increased, the geometry of the junction becomes less complex.
Although there is the possibility of some new strand material being formed after D.C treatment, the general response of the junction to 1 hour of either specific treatment is a rearrangement of existing strand material and this is most readily detected by analysis using method 5.
References.
i. 2. 3. 4. 5. 6. 7. 8.
Claude, P. & Goodenough, D.A. 1973. J.Cell Biol. 58, 390-400. Bullivant, S. 1981. 'Epithelial Ion & Water transport.' Ed. Macknight, Raven Press, N . Y . p . 2 6 5 - 2 7 5 . Riddle, C.V. & Ernst, S.A. 1979. J. Memb. Biol. 45, 21-35. Wade, J.B. & Karnovsky, M.J. 1974. J. Cell Biol. 62, 344-350 Bindslev, N. et a 1 1 9 7 4 . Biochim. Biophys. Acta. 33__2, 286-297. Joyoe, P.R. et a 1 1 9 7 6 . Proc. Univ. Otago Med. Sch. 5_44. 47-48. Murphy, C.R. et al. 1981. Cell Biophys. ~, 57-69. Hull, B.E., Staehelin L.A. 1976. J. Cell Biol. 68. 688-704.
Acknowledgement. The authors thank the Medical Research Council of N.Z. for financial support.