가재미 단세요관(單細尿管) 표준에서의 phenol red 유도체의 분비기전(分泌機轉)에 관하여
Since Marshall and Vickers established in 1923 that the renal tubule is able to secrete certain dyes such as phenol red, many investigators worked on the problem of tubular secretory process. Thus, it is now widely accepted that the tubular secretion is carried out by an active transport system which requires supply of metabolic energy and is also subject to competitive inhibition. However, the process of tubular secretion at the cellular level was not well understood until Forster(1948) developed a technique in which the transport of a dye can be studied in isolated renal tublue preparations by direct visualization. Using this method, Forster and Taggart (1950) characterized the nature of active transport as well as of competitive inhibition. In addition. Puck et al(1952) skewed that the transfer of phenol red from the medium to the cell (Step Ⅰ) requires K**+ while that from the cell to the lumen (Step Ⅱ) requires Ca**++. Forster and Hong (1958) worked on the tubular transport of several other phenol red derivatives and reported that chlorphenol red (CPR) is secreted by the same manner as phenol red while bromphenol blue (BPB) and bromcresol green (BCG) are transported in a manner different from phenol red. However, more extensive studies are needed in characterizing the differences in the process of tubular secretion between CPR and BPB (or BCG).
In 1958, Heng and Forater has been able to show that CPR preaccumulated in the tubular lumen by the uptake process (i.e., secretion) is subject to back-diffusion which they designated as "run-out" process. These authors analyzed this run-out process in detail and then characterized the permeability characteristics to CPR of the peritubular membrane as well as of the luminal side of the membrane. Recently, Hinter and Cline (1961) also showed that Diodrast preaccumulated in the tubule (both the cell and the lumen) is also subject to run-out, but that this run-out process is carried out by "facilitated diffusion" coupled with "exchange
On the oasis of these considerations, the present investigation was undertaken to study 1) both uptake and run-out processes of BPB and BCG under various conditions and 2) extend earlier studios on run-out of CPR in the light of a new theory by
Hinter and Cline.
Experiments were carried out in the isolated renal tubules of 145 flounders which were approximately 5 inches in length. The flounder was caught by fishing at Masan seashore and the experiment was started within 15 minutes. The preparation of the
isolated . renal tubule was similar to that described by Forster and Hong (1958).
An incision wag made through the vertebral column in the area of the gills, and within several seconds small fragments of renal tissue were transferred to an oxygenated, balanced, isotonic salt solution (Forster medium) which had been empirically derived for its ability to sustain secretory activity in renal tubules.
This solution contained the following concentration of salts in millimoles per liter: NaCI 135, CaCI^^2 1.5, KCI 2.5, MgCl^^2 1.0, NaH^^2 PO^^4 0.5, and NaHCO^^3 10.0. Petri dishes (1.2×4.0 cm) containing 5 to 7ml of the sustaining medium were used to observe dye transport in teased kidney fragments under 100x magnification.
Usually 6 such Petri dishes, containing 3× 10**-5 M concentration of the various dyes in the medium were oxygenated simultaneously via 22 gauge hypodermic needles at laboratory temperature. Oxygenation was interrupted momentarily when dishes were transferred to the microscope stage from time to time for evaluation of dye concentration in cells or lumina.
The uptake study was carried out for a period of 60 minutes during which the degree of uptake was evaluated every 20 minutes. For the run-out study, the dye was preaccumulated in the tubule for a period of 40 to 60 minutes and then the preparation wag transferred to oxygenated, dye-free Forster medium after which the degree of run-out was evaluated every 20 minutes for 60 minutes. In those experiments in which the effects of metabolic inhibitors or competitive inhibitors on the tubular transport were studied, an inhibitor was added to the Forster medium. Moreover, a certain electrolyte such as K**+ or Ca**++ was removed from the Forster medium when the effect of respective ion on the tubular transport was studied. The evaluation of dye concentration in cells and lumina was based on a semiquatitative method of Forater and Hong (1958) who assigned arbitrary concentration ratings ranging from+(definitely detectable) to++++(maximal) in accordance with the behavior of the majority of tubules.
The following series of experiments were carried out:
A. Normal uptake and run·out of the dye (16 flounders).
B. Effects of K**+ and Ca**++ on uptake and run-out of the dye (24 flounders).
C. Effects of competitive inhibitors on uptake and run-out of the dye.
(1) Para-aminohippuric acid (PAH) (13 flounders).
(2) Benemid (12 flounders).
(3) Diodrast (15 flounders).
D. Effects of metabolic inhibitors on uptake and run-out of the dye.
(1) 2, 4 dinitrophenol (DNP) (16 flounders).
(2) Sodium cyanide (NaCN) (14 flounders).
E. Run-out of the dye, preaccumulated in presence of NaCN, in Ca**++-free and dye-free medium (5 flounders).
F. Run-out of chlorphenol red in presence of competitive inhibitors of varying concentration.
(1) Benemid (7 flounders).
(2) PAH (8 flounders).
(3) DNP and Benemid (8 flounders).
(4) DNP and PAH (7 flounders).
Results may be briefly summarized as follows:
1. BPB and BCG were accumulated homogeneously in both the tubular lumen and the cell, while CPR was accumulated only in the tubular lumen.
2. As in the case of CPR, BPB and BCG were also accumulated only in the cell in Ca**++ free medium.
3. In Ca**++ free and dye free medium, run-out of BPB and BCG was facilitated as in the casts of CPR.
4. In K**+ free medium, CPR was not accumulated in either the tubular lumen or the cell. However, BPB and BCG were accumlated homogeneously in both the lumen and the cell, although the concentration was lower than normal.
5. In K**+ free and dye free medium, run-out was not modified in all dyes.
6. Competitive inhibitors such as PAH, Benemid and Diodrast had no effect on either uptake or run-out of BPB and BCG, while uptake of CPR was inhibited and run-out was facilitated by these inhibitors.
7. DNP had no effect on uptake and run-out of BPB and BCG at a concentration of 10**-4 M, but facilitated run-out without any effect on uptake at a concentration of 10**-3 M.
8. In presence of 10**-3 M of NaCN in the medium, BPB and BCG were accumulated only intracellularly: moreover, in the process of run-out, these dyes were accumulated only in the cell, and intracellularly accumulated dye did net run out even in Ca**++-free and dye-free medium.
9. Although Benemid facilitated run-out of CPR at a concentration below 10**-3 M, it inhibited at 10**-2 M.
10. PAH facilitated run-out of CPR at all concentrations ranging from 10**-5 to 10**-2 M.
11. In presence of both DNP and a competitive inhibitor, run-out of CPR was facilitated to the same extent as with DNP alone.
These results indicate that the transport process of BPB and BCG is quite different from that of CPR, although all these dyes are phenol red derivatives and thus one would expect a great deal of similarities in transporting them across the renal tubule. However, the reason (s) for this marked difference is not clear at present. Moreover, the results on run-out of CPR in the medium containing various concentrations of Benemid suggests that the run-out process is more than a simple back-diffusion.