- Email: [email protected]
Chapter 10 Changes in Cell Membrane Surfaces Associated with Alterations of Transepithelial Ion Movement
CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. VOLUME
13
Chapter 10 Changes in Cell Membrane Surfaces Associated with Alterations of Transepithelial Ion Movement MICHAEL KASHGARIAN Department of Pathology Yale University School of Medicine Nen. Hnven, Connecticut
. . . . . 111. Colon Structure . . . IV. Renal Structure . . .
. . . . . . . . . . . . . V . Structure-Function Correlations . . . References . . . . . . . . . . . I.
. . . . . . . .
Introduction
11. Stereologic Methods .
1.
. . . . . . . . . . . .
. . . . . . . . . . . . . . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
149
. 150
. . I51 . . 155 . . 156 . 159
INTRODUCTION
Vertebrates in their evolution have developed sophisticated means to maintain and regulate their internal osmotic and ionic environment. In vertebrates, a variety of organ systems are involved in osmoregulation and their relative importance varies between phyla, classes, orders, genera, and even species (2). The organ systems of importance include the skin, gills, lungs, gut, urinary bladder, specialized salt glands, and kidney. This multiplicity of osmoregulatory organs allows for a variety of mechanisms to adapt to significant changes in the environment. For example, euryhaline teleosts, particularly anandromous fish, adapt to varying degrees of salinity with changes in renal, gill, and gut function (4, 8). Such adaptive changes are controlled or mediated by alterations in endocrine function and a variety of hormonal systems appear to contribute to adaptation ( 2 ) . In mammals, the internal osmotic and ionic environment is regulated by a series of complex and efficient mechanisms which maintain low 149
150
MICHAEL KASHGARIAN
concentrations of potassium outside the cell and high concentrations of potassium in the intracellular fluid. This special environment is defended to narrow limits despite wide variations of dietetic intake and pathological alterations inhibiting excretion. For example, animals receiving increasing amounts of potassium in their diet become resistant to loads of potassium which would normally be lethal in unadapted animals (3, 19). Such animals excrete large amounts of potassium even when not challenged by acute potassium loads and have an enhanced ability to excrete a sudden challenge of excessive potassium. This enhanced excretion occurs since both the kidney and the gut respond to such potassium adaptation with an enhanced ability to secrete potassium in the urine and the stool (7, 1 5 ) . Na+- K+-ATPase appears to be responsible for the peritubular uptake of potassium in exchange for intracellular sodium and might therefore be involved in any adaptive regulatory mechanisms. A selective increase of this enzyme occurs in the renal tubular cells in potassium adaptation initially in the outer medulla and, after further increases in dietary potassium, in the cortex as well (5, 17). The colonic epithelium similarly responds to chronic potassium loading with a selective increase in Na+K+-ATPase (6, 16). Mineralocorticoids appear to be able to mimic the effects of chronic potassium loading functionally in relationship to the ability to excrete acute potassium loads (10, 19). This is accompanied by increases in Na+K+-ATPase in the kidney similar to the effect of chronic potassium loading. In the colon, glucocorticoids also appear to have similar effects (1). There is increased fluid and sodium absorption as well a s an increase in transepithelial potential and Na+- K+-ATPase activity of the epithelium (6).
Since physiological adaptation to different ionic environments appears to be accompanied by changes in the activity of an enzyme which is closely bound to cell membranes, studies were undertaken to assess whether such adaptive changes in potassium secretion, sodium reabsorption, and enzyme activity were accompanied by structural changes in the cell membrane surfaces. In order to avoid errors in interpretation due to sampling techniques, quantitative stereologic methods were used to evaluate membrane surface density and its relationship to any changes or differences in cell size and epithelial exchange surface. II. STEREOLOGIC METHODS
Stereologic techniques allow for morphometric quantitation of threedimensional objects through the analysis of two-dimensional electron micrographs (20). Quantitative relationships exist between the average
151
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
dimension of profiles of a particular structure of section to the surface areas of the same structure in its three-dimensional reality. This relationship is given by the equation B, = (7r/4) s,,.The length of profile borders can be estimated by placing a grid of test lines separated by a known distance, d , on the electron micrograph and counting the intersections, i , formed by these lines with the profile border. The border, B , , is given by the equation B, = (7r/2) x i x d and it is immediately seen that substitution of the second equation into the first yields a direct relationship between the surface density s,, and the number of intersections. These techniques require the sampling of cells from individual animals and from groups of animals in different physiologic states. For the purposes of evaluating the structural changes seen with potassium adaptation in the colon four groups of animals were studied (18). In the first, rats were fed supplemental potassium equivalent to 50 mEq of potassium chloride per day for 1 week. The second group received the glucocorticoid. dexamethasone (600 pgl100 gm body wt/day) for 3 days. The third group was chronically depleted of sodium by maintenance on a sodium-free diet for a period of 2 weeks. The last group was a control group of littermates fed a normal diet containing 5 mEq of potassium per day prior to sacrifice. After anesthesia the colon was fixed by arterial perfusion with dilute Karnovsky's solution. Since functional differences in some transport properties have been demonstrated in the proximal and distal colon, tissue samples were taken from each segment in control and experimental groups. After routine processing for electron microscopy, electron micrographs containing parallel arrays of absorptive cells with visible nuclei, intact basement membranes, and abundant microvilli were selected for measurement and were individually coded. The coded electron micrographs were measured by an observer unaware of the code and therefore the pretreatment group. Using standard stereologic techniques the membrane surface density S , of the basolateral cell surfaces was measured. The membrane surface density is the surface-to-volume ratio determined from the stereologic techniques described earlier. The surface concentration, S,, of the microvilli on the luminal surface was also measured. This parameter is a ratio of the villous surface area to a square millimeter of plane cell surface and thus represents the factor of multiplication of the plane cell surface by the microvilli. 111.
COLON STRUCTURE
Striking differences were found between the cells of the proximal and distal colon. The cells of the proximal colon were taller and wider than the cells of the distal colon measuring 36.8 I . 1 F m in height and 5.5 2
*
152
MICHAEL KASHGARIAN
FIG. I . Electron micrographs of proximal and distal colonic epithelium of a control rat. The micrographs are at exactly the same magnification ( x 3000). The proximal colon on the left has taller and wider cells than the distal colon. Although the membrane surface concentration is greater in the distal colon (see text) the proximal colon has more total membrane area.
0.2 p m in width as compared with 21.4 f 0.8 p m height and 4.0 k 0.2 p m width of the epithelial cells of the distal colon (Fig. I ) . The basolateral membrane surface concentration was greater in the distal colon than in the proximal, being 1.75 f 0.06 pm2/pm3 as compared to 1.40 f 0.05 pm2/pm3 in the proximal colon. The difference persists even if the surface concentration is corrected to compensate for the differences in cell width h relating S , to a unit length of basement membrane which can be regarded as an index of available epithelial exchange surface by dividing S , by the cell width. The microvillous surface concentration S, is also greater in the distal colon measuring 33.2 k 1. I pm2/pm2as compared to 19.3 h I .5 pm2/prn2.These structural differences have parallel functional
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
153
FIG. 2 . Scanning electron micrograph of the lateral surfaces of colon epithelial cells. Only lateral interdigitations are near the apex of the cells. At the base of the cells the interdigitations involve both basal and lateral portions of the cells. ~ 3 9 0 0 .
differences. A variety of studies have demonstrated that the proximal colon absorbs more sodium, has a greater passive permeability, and a lower transepithelial potential than the distal colon. The distal colon is capable of maintaining greater ionic gradients and has a higher transmural potential difference. In the group of animals fed excess potassium chloride for a week previous studies have demonstrated that physiological adaptation occurs with increased secretion of potassium and an increased capability of handling acute potassium loads. Electron microscopy of the colonic cells demonstrates that the intercellular surfaces are more complex when compared to normal. Scanning electron microscopy of the lateral surfaces more vividly demonstrates the nature of the interdigitations (Fig. 2). Lateral interdigitations are most prominent near the apex of the cell while more classical basolateral interdigitations are present near the base. The subjective changes seen on the electron micrographs were quantitated by the stereologic techniques. A difference between the proximal and distal colon persists and an increase in basolateral surface density is observed in both segments of the colon (Figs. 3 and 4). In the proximal colon there was a 39% increase with the surface density of potassium-adapted rats
154
MICHAEL KASHGARIAN
FIG.3. Electron micrograph of intercellular junctions and interdigitations of distal colon epithelial cells from a control rat. The interdigitations are relatively simple. X 14,600.
measuring 1.85 +- 0.06 pm2/pm3as compared to the control value of 1.40 I+- 0.05. In the distal colon there was an increase of 51% with a basolateral surface density measuring 2.80 f 0.05 pm2/pm3 as compared to the control value of 1.75 f 0.05. Microvillous surface concentration, however, did not change when compared to control measuring 20.3 & 2.1 pm2/pm2 in the proximal colon and 32.6 1.2 pm2/pm2 in the distal colon. Treatment with dexamethasone for 3 days also resulted in an increase in membrane surface density (Fig. 5). This could be appreciated in electron micrographs as well as with the stereologic techniques. The quantitative change was not as striking as seen with potassium adaptation but this is probably related to the short duration of dexamethasone treatment and thus probably represents an early stage of membrane formation. There was a change of 28% in the proximal colon with a basolateral surface density measuring 1.32 0.75 pm2/pm3and an increase of 16% in the distal colon with a basolateral surface density of 2.15 +- 0.10 pm2/ pm3. Chronic sodium depletion is sufficient to induce an endogenous secretion of the mineralocorticoid aldosterone resulting in a marked increase in the basolateral surface density in a pattern similar to that seen with dexamethasone treatment with a 71% increase in the proximal colon _+
_+
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
155
FIG.4. Electron micrograph of intercellular junctions and interdigitations from a potassium-adapted rat. The complexity of the interdigitations is markedly increased. The membrane surface density (S,)reflects this change. x 18,300.
with the basolateral surface density measuring 2.40 & 0.1 and a 56%' increase in the distal colon with a basolateral surface density of 2.92 2 0.1 pm2/pm3.
IV.
RENAL STRUCTURE
As was discussed previously, potassium adaptation occurs in many organ systems, and in the kidney functional and biochemical changes, similar to those of the colon, occur in the distal tubule. Recent microcatheterization studies by Schon and Hayslett (14) have demonstrated an adaptive potassium secretion in the medullary collecting duct. In addition, the increase in Na+- K+-ATPase levels particularly in the outer medulla associated with potassium adaptation as well as the physiological studies performed by others (15, 17) have localized the distal portions of the nephron as being the most important to be effected by potassium adaptation. Because of the physiologic similarities between the colon and the distal nephron, it was important to determine whether an increase in basolateral membrane surface was an inherent component of the compensatory adaptation to potassium secretion.
MICHAEL KASHGARIAN
156
FIG. 5 . Electron micrograph of intercellular interdigitations of a rat treated with the glucocorticoid dexamethasone. The increase in basolateral membranes is similar to that seen with potassium adaptation. ~ 8 , 6 7 0 .
Quantitative stereologic studies of collecting ducts of the outer medulla were performed (/.<). The kidneys were fixed by retrograde perfusion with dilute Karnovsky's fixative and prepared for electron microscopy using standard techniques. Using quantitative stereologic techniques similar to those used in the colon, the basolateral surface density was measured. The difference in basolateral membrane surfaces can easily be appreciated by a subjective examination of electron' micrographs (Figs. 6 and 7). There is an increase in the surface area and in the complexity of these interdigitations in the potassium adaptation. The quantitative stereologic studies demonstrated a 43% increase in basolateral membrane surface density with potassium-adapted animals having an S, of 2.70 & 0.24 pm2/ pm3 as compared to the control value of 1.90 & 0.12 pm2/pm3. This change is similar to that seen in the colon. Wade (Chapter 9, in this volume) has also demonstrated an increase in basolateral surface density in isolated cortical collecting tubules of rabbits treated with DOCA. V.
STRUCTURE-FUNCTION CORRELATIONS
It is apparent, therefore, that in the mammalian tissues studied the
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
157
FIG.6. Electron micrograph of basolateral interdigitations of outer medullary collecting duct cells of a control rat. The moderately complex lateral membrane system can be easily appreciated. X 12,900.
physiological and biochemical changes induced by alterations in the ionic environment or through hormonal mediation are accompanied by striking structural changes reflected in an increase in the basolateral membrane surface. There are at least two significant physiological implications of such structural changes. The increase in basolateral membrane surface density may be related to an increase in the number of available transport sites. Evidence for this is suggested by the parallel increase in Na+-K+ATPdse in the adapted tissues. For example, the 43% increase in membrane surface density seen in the outer medullary collecting tubule in potassium adaptation roughly corresponds to the 35% increase in Na+K+-ATPase activity of the outer medullary portion of the kidney (17). In the colon, although the 2-fold increase in enzyme activity which has been observed (6) is not quite matched by the increase in membrane surface density, the direction of change is the same and the difference may be accounted for by a heterogeneity of the tissue utilized for enzyme analysis. A parallel appears to be present in nonmammalian tissues. The euryhaline teleost Gristcwsfws t r c w l m f n demonstrates striking changes in the basolateral membrane of the renal tubule during adaptation from fresh
158
MICHAEL KASHGARIAN
FIG.7. Electron micrograph of basolateral interdigitations of outer medullary collecting duct of a potassium-adapted rat. The complexity of the lateral membranes is markedly increased. This is reflected in the increase in S, determined stereologically. x 12,500.
water to sea water (21). A WO increase in basolateral membrane surface density has been observed and this appears to correspond to the increase in ion absorption as reflected by a lowering of the urine osmolarity during such adaptation. Although no enzyme analyses have been carried out in this particular species of fish, in other euryhaline teleosts such as the killifish, adaptation to fresh water is accompanied by an increase in Na+K+-ATPase of renal tissue (12). Similar qualitative structural changes have also been observed in the opposite direction in the renal tubule of the rainbow trout, Snlrno griirdneri, when such fish are adapted from fresh water to salt water ( / I ) .The nasal salt glands of aquatic birds also appear to undergo similar changes in both enzymatic activity and membrane structure during changes of salt intake ( 9 ) . It should also be noted that such an increase in the basolateral membrane surface area is accompanied by an increase in the length and complexity of the intercellular spaces. The importance of the paracellular pathway for transepithelial fluid flow is discussed in other articles in this volume. The structural changes observed here could alter intercellular osmotic gradients o r the paracellular conductance which could in turn have major effects on net transepithelial movement of ions and fluid. It
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
159
is thus apparent that the structural changes associated with ionic adaptation could affect transepithelial ionic movement either as a result of changes in some active component such as in transport enzyme sites o r through alterations of passively induced osmotic fluid flow through the intercellular channels. These observations lead to yet another area of speculation in relation to the mechanism of induction of these membrane changes. In the studies described here, membrane changes were associated with changes in potassium intake, glucocorticoid levels, and mineralocorticoid levels. I t would be interesting if a single effector mechanism could account for all of these environmental changes. Studies in teleosts have suggested that the anterior pituitary hormone, prolactin, appears to be the effector hormone responsible for control of adaptation to fresh water. Its importance has been demonstrated in the killifish. F/rndrr/ir.shc~to.oc/itri.c (/,?), and several species of salmon and trout ( 2 , 8). Furthermore, in studies of the stickleback fish, the structural changes of ionic adaptation have been accelerated as well as duplicated by the action of prolactin (22). Since in humans sodium depletion and steroid therapy are associated with an increase in serum prolactin, it is interesting to speculate that this hormone may indeed be a common mediator involved in the structural and functional adaptations described here. REFERENCES I . Bastl, C. P., Hayslett. J . P., and Binder, H. J . (1977). Role of glucocorticoids on ion transport and transmural potential difference in colon. Cliii. Reg. 25, 306. 2. Bentley. P. J. ( 1971). "Endocrines and Osmoregulation." Springer-Verlag, Berlin and
New York. 3 . Berliner. R. W . , Kennedy, T. G . , and Hilton. J . 0.(1950). Renal mechanisms for excretion of potassium. A m . J. Physiol. 162, 348-367. 4. Ensor. D. M., and Ball, J. N. (1972). Prolactin and osmoregulation. Fed. Pro(,. Fed. A i i i . Soc. E.vp. B i d . 31, 1615-1623. 5 . Finkelstein, F. 0..and Hayslett, J . P. (1975). Role of medullary Na-K ATPase in renal l . S74-528. potassium adaptation. A m . J. P h y ~ i ~229, 6. Fisher, K., Binder, H . J . , and Hayslett. J . P. (1976). Potassium secretion by colonic mucosal cells after potassium adaptation. A m . J . Physiol. 231, 987-994. 7. Hayes, C. P., McLeod, M . E., and Robinson, R. R . (1967). An extrarenal mechanism for the maintenance of potassium balance in severe chronic renal failure. 7'nrri.s. Assoc. A m . Physician.c 80, 207-216. 8 . Hoar, W . S . , and Randall, D. J . (1969). "Fish Physiology." Academic Press, New York. 9. Holmes, W . N. (1972). Regulation of electrolyte balance in marine birds. Fed. Proc. Fod. A m . Soc. E x p . B i d . , 31, 1587-1598. 10. Jorgensen, P. L. (1969). Regulation of Na-K ATPase in the rat kidney. 11. The effect of adolsterone on the activity in kidneys of adrenalectomized rat. Biochini. Biophy.~. Acrcc 192, 326-334.
160
MICHAEL KASHGARIAN
II, Kashgarian, M . (1977). Unpublished observations. 12. Pickford, G. E., Griffith, R. W., Torretti, J., Hendler, E., and Epstein, F. H. (1970).
13. 14. 15.
16. 17.
18. 19. 20.
21. 22.
Branchial reduction and renal stimulation of Na-K ATPase by prolactin in hypophysectomized killifish in fresh water. Nuture (London) 228, 378. Rastegar, A., Hayslett, J . P., and Kashgarian, M . (1980). Changes in membrane surfaces of collecting duct cells in potassium adaptation. Kidney I n r . (in press). Schon, D. A., and Hayslett, J . P. (1978). Potassium adaptation in medullary collecting duct. Kidney I n r . 14, 780. Schon, D . A., Silva, P., and Hayslett, J. P. (1974). The mechanism of renal potassium excretion in uremia. A m . J . Physiol. 227, 1323-1330. Silva, P., Charney, A. N., and Epstein, F. H. (1975). Potassium adaptation and Na-K ATPase activity in mucosa of colon. A m . J . Physiol. 22Y, 1576-1579. Silva, P., Hayslett, J . P., and Epstein, F. H. (1973). The role of Na-K ATPase in potassium adaptation. J . C ' h . lnves/. 52, 2665-267 I . Taylor, C. R., Hayslett, J . P., and Kashgarian, M. (1978). Structural adaptation of cell membrane surfaces to compensatory increases in potassium secretion. Proc. lnr. C'ongr. Neplzrol., 7/11 C9. Thatcher, J . S . , and Radike, A. W. (1947). Tolerance to potassium intoxication in the albino rat. Am. J . Physiol. 151, 138-146. Weibel, E. R. ( 1969). Stereological principles for morphometry in electron microscopic cytology. I n ! . R e v . Cytol. 26, 335. Wendelaar Bonga, S. E., and Veenhuis, M . (1974). The membranes of basal labyrinth in kidney cells in the anandromous teleost Gusterosreus nculearu in adaptation from sea water to fresh water. J . Cell Sci. 14, 587-609. Wendelaar Bonga, S . E. ( 1976). The effect of prolactin on kidney structure of euryhaline teleost Gusterosteus uculrtrrci during adaptation to fresh water. Cell 7issue Rev. 166, 3 19-338.
13
Chapter 10 Changes in Cell Membrane Surfaces Associated with Alterations of Transepithelial Ion Movement MICHAEL KASHGARIAN Department of Pathology Yale University School of Medicine Nen. Hnven, Connecticut
. . . . . 111. Colon Structure . . . IV. Renal Structure . . .
. . . . . . . . . . . . . V . Structure-Function Correlations . . . References . . . . . . . . . . . I.
. . . . . . . .
Introduction
11. Stereologic Methods .
1.
. . . . . . . . . . . .
. . . . . . . . . . . . . . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
149
. 150
. . I51 . . 155 . . 156 . 159
INTRODUCTION
Vertebrates in their evolution have developed sophisticated means to maintain and regulate their internal osmotic and ionic environment. In vertebrates, a variety of organ systems are involved in osmoregulation and their relative importance varies between phyla, classes, orders, genera, and even species (2). The organ systems of importance include the skin, gills, lungs, gut, urinary bladder, specialized salt glands, and kidney. This multiplicity of osmoregulatory organs allows for a variety of mechanisms to adapt to significant changes in the environment. For example, euryhaline teleosts, particularly anandromous fish, adapt to varying degrees of salinity with changes in renal, gill, and gut function (4, 8). Such adaptive changes are controlled or mediated by alterations in endocrine function and a variety of hormonal systems appear to contribute to adaptation ( 2 ) . In mammals, the internal osmotic and ionic environment is regulated by a series of complex and efficient mechanisms which maintain low 149
150
MICHAEL KASHGARIAN
concentrations of potassium outside the cell and high concentrations of potassium in the intracellular fluid. This special environment is defended to narrow limits despite wide variations of dietetic intake and pathological alterations inhibiting excretion. For example, animals receiving increasing amounts of potassium in their diet become resistant to loads of potassium which would normally be lethal in unadapted animals (3, 19). Such animals excrete large amounts of potassium even when not challenged by acute potassium loads and have an enhanced ability to excrete a sudden challenge of excessive potassium. This enhanced excretion occurs since both the kidney and the gut respond to such potassium adaptation with an enhanced ability to secrete potassium in the urine and the stool (7, 1 5 ) . Na+- K+-ATPase appears to be responsible for the peritubular uptake of potassium in exchange for intracellular sodium and might therefore be involved in any adaptive regulatory mechanisms. A selective increase of this enzyme occurs in the renal tubular cells in potassium adaptation initially in the outer medulla and, after further increases in dietary potassium, in the cortex as well (5, 17). The colonic epithelium similarly responds to chronic potassium loading with a selective increase in Na+K+-ATPase (6, 16). Mineralocorticoids appear to be able to mimic the effects of chronic potassium loading functionally in relationship to the ability to excrete acute potassium loads (10, 19). This is accompanied by increases in Na+K+-ATPase in the kidney similar to the effect of chronic potassium loading. In the colon, glucocorticoids also appear to have similar effects (1). There is increased fluid and sodium absorption as well a s an increase in transepithelial potential and Na+- K+-ATPase activity of the epithelium (6).
Since physiological adaptation to different ionic environments appears to be accompanied by changes in the activity of an enzyme which is closely bound to cell membranes, studies were undertaken to assess whether such adaptive changes in potassium secretion, sodium reabsorption, and enzyme activity were accompanied by structural changes in the cell membrane surfaces. In order to avoid errors in interpretation due to sampling techniques, quantitative stereologic methods were used to evaluate membrane surface density and its relationship to any changes or differences in cell size and epithelial exchange surface. II. STEREOLOGIC METHODS
Stereologic techniques allow for morphometric quantitation of threedimensional objects through the analysis of two-dimensional electron micrographs (20). Quantitative relationships exist between the average
151
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
dimension of profiles of a particular structure of section to the surface areas of the same structure in its three-dimensional reality. This relationship is given by the equation B, = (7r/4) s,,.The length of profile borders can be estimated by placing a grid of test lines separated by a known distance, d , on the electron micrograph and counting the intersections, i , formed by these lines with the profile border. The border, B , , is given by the equation B, = (7r/2) x i x d and it is immediately seen that substitution of the second equation into the first yields a direct relationship between the surface density s,, and the number of intersections. These techniques require the sampling of cells from individual animals and from groups of animals in different physiologic states. For the purposes of evaluating the structural changes seen with potassium adaptation in the colon four groups of animals were studied (18). In the first, rats were fed supplemental potassium equivalent to 50 mEq of potassium chloride per day for 1 week. The second group received the glucocorticoid. dexamethasone (600 pgl100 gm body wt/day) for 3 days. The third group was chronically depleted of sodium by maintenance on a sodium-free diet for a period of 2 weeks. The last group was a control group of littermates fed a normal diet containing 5 mEq of potassium per day prior to sacrifice. After anesthesia the colon was fixed by arterial perfusion with dilute Karnovsky's solution. Since functional differences in some transport properties have been demonstrated in the proximal and distal colon, tissue samples were taken from each segment in control and experimental groups. After routine processing for electron microscopy, electron micrographs containing parallel arrays of absorptive cells with visible nuclei, intact basement membranes, and abundant microvilli were selected for measurement and were individually coded. The coded electron micrographs were measured by an observer unaware of the code and therefore the pretreatment group. Using standard stereologic techniques the membrane surface density S , of the basolateral cell surfaces was measured. The membrane surface density is the surface-to-volume ratio determined from the stereologic techniques described earlier. The surface concentration, S,, of the microvilli on the luminal surface was also measured. This parameter is a ratio of the villous surface area to a square millimeter of plane cell surface and thus represents the factor of multiplication of the plane cell surface by the microvilli. 111.
COLON STRUCTURE
Striking differences were found between the cells of the proximal and distal colon. The cells of the proximal colon were taller and wider than the cells of the distal colon measuring 36.8 I . 1 F m in height and 5.5 2
*
152
MICHAEL KASHGARIAN
FIG. I . Electron micrographs of proximal and distal colonic epithelium of a control rat. The micrographs are at exactly the same magnification ( x 3000). The proximal colon on the left has taller and wider cells than the distal colon. Although the membrane surface concentration is greater in the distal colon (see text) the proximal colon has more total membrane area.
0.2 p m in width as compared with 21.4 f 0.8 p m height and 4.0 k 0.2 p m width of the epithelial cells of the distal colon (Fig. I ) . The basolateral membrane surface concentration was greater in the distal colon than in the proximal, being 1.75 f 0.06 pm2/pm3 as compared to 1.40 f 0.05 pm2/pm3 in the proximal colon. The difference persists even if the surface concentration is corrected to compensate for the differences in cell width h relating S , to a unit length of basement membrane which can be regarded as an index of available epithelial exchange surface by dividing S , by the cell width. The microvillous surface concentration S, is also greater in the distal colon measuring 33.2 k 1. I pm2/pm2as compared to 19.3 h I .5 pm2/prn2.These structural differences have parallel functional
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
153
FIG. 2 . Scanning electron micrograph of the lateral surfaces of colon epithelial cells. Only lateral interdigitations are near the apex of the cells. At the base of the cells the interdigitations involve both basal and lateral portions of the cells. ~ 3 9 0 0 .
differences. A variety of studies have demonstrated that the proximal colon absorbs more sodium, has a greater passive permeability, and a lower transepithelial potential than the distal colon. The distal colon is capable of maintaining greater ionic gradients and has a higher transmural potential difference. In the group of animals fed excess potassium chloride for a week previous studies have demonstrated that physiological adaptation occurs with increased secretion of potassium and an increased capability of handling acute potassium loads. Electron microscopy of the colonic cells demonstrates that the intercellular surfaces are more complex when compared to normal. Scanning electron microscopy of the lateral surfaces more vividly demonstrates the nature of the interdigitations (Fig. 2). Lateral interdigitations are most prominent near the apex of the cell while more classical basolateral interdigitations are present near the base. The subjective changes seen on the electron micrographs were quantitated by the stereologic techniques. A difference between the proximal and distal colon persists and an increase in basolateral surface density is observed in both segments of the colon (Figs. 3 and 4). In the proximal colon there was a 39% increase with the surface density of potassium-adapted rats
154
MICHAEL KASHGARIAN
FIG.3. Electron micrograph of intercellular junctions and interdigitations of distal colon epithelial cells from a control rat. The interdigitations are relatively simple. X 14,600.
measuring 1.85 +- 0.06 pm2/pm3as compared to the control value of 1.40 I+- 0.05. In the distal colon there was an increase of 51% with a basolateral surface density measuring 2.80 f 0.05 pm2/pm3 as compared to the control value of 1.75 f 0.05. Microvillous surface concentration, however, did not change when compared to control measuring 20.3 & 2.1 pm2/pm2 in the proximal colon and 32.6 1.2 pm2/pm2 in the distal colon. Treatment with dexamethasone for 3 days also resulted in an increase in membrane surface density (Fig. 5). This could be appreciated in electron micrographs as well as with the stereologic techniques. The quantitative change was not as striking as seen with potassium adaptation but this is probably related to the short duration of dexamethasone treatment and thus probably represents an early stage of membrane formation. There was a change of 28% in the proximal colon with a basolateral surface density measuring 1.32 0.75 pm2/pm3and an increase of 16% in the distal colon with a basolateral surface density of 2.15 +- 0.10 pm2/ pm3. Chronic sodium depletion is sufficient to induce an endogenous secretion of the mineralocorticoid aldosterone resulting in a marked increase in the basolateral surface density in a pattern similar to that seen with dexamethasone treatment with a 71% increase in the proximal colon _+
_+
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
155
FIG.4. Electron micrograph of intercellular junctions and interdigitations from a potassium-adapted rat. The complexity of the interdigitations is markedly increased. The membrane surface density (S,)reflects this change. x 18,300.
with the basolateral surface density measuring 2.40 & 0.1 and a 56%' increase in the distal colon with a basolateral surface density of 2.92 2 0.1 pm2/pm3.
IV.
RENAL STRUCTURE
As was discussed previously, potassium adaptation occurs in many organ systems, and in the kidney functional and biochemical changes, similar to those of the colon, occur in the distal tubule. Recent microcatheterization studies by Schon and Hayslett (14) have demonstrated an adaptive potassium secretion in the medullary collecting duct. In addition, the increase in Na+- K+-ATPase levels particularly in the outer medulla associated with potassium adaptation as well as the physiological studies performed by others (15, 17) have localized the distal portions of the nephron as being the most important to be effected by potassium adaptation. Because of the physiologic similarities between the colon and the distal nephron, it was important to determine whether an increase in basolateral membrane surface was an inherent component of the compensatory adaptation to potassium secretion.
MICHAEL KASHGARIAN
156
FIG. 5 . Electron micrograph of intercellular interdigitations of a rat treated with the glucocorticoid dexamethasone. The increase in basolateral membranes is similar to that seen with potassium adaptation. ~ 8 , 6 7 0 .
Quantitative stereologic studies of collecting ducts of the outer medulla were performed (/.<). The kidneys were fixed by retrograde perfusion with dilute Karnovsky's fixative and prepared for electron microscopy using standard techniques. Using quantitative stereologic techniques similar to those used in the colon, the basolateral surface density was measured. The difference in basolateral membrane surfaces can easily be appreciated by a subjective examination of electron' micrographs (Figs. 6 and 7). There is an increase in the surface area and in the complexity of these interdigitations in the potassium adaptation. The quantitative stereologic studies demonstrated a 43% increase in basolateral membrane surface density with potassium-adapted animals having an S, of 2.70 & 0.24 pm2/ pm3 as compared to the control value of 1.90 & 0.12 pm2/pm3. This change is similar to that seen in the colon. Wade (Chapter 9, in this volume) has also demonstrated an increase in basolateral surface density in isolated cortical collecting tubules of rabbits treated with DOCA. V.
STRUCTURE-FUNCTION CORRELATIONS
It is apparent, therefore, that in the mammalian tissues studied the
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
157
FIG.6. Electron micrograph of basolateral interdigitations of outer medullary collecting duct cells of a control rat. The moderately complex lateral membrane system can be easily appreciated. X 12,900.
physiological and biochemical changes induced by alterations in the ionic environment or through hormonal mediation are accompanied by striking structural changes reflected in an increase in the basolateral membrane surface. There are at least two significant physiological implications of such structural changes. The increase in basolateral membrane surface density may be related to an increase in the number of available transport sites. Evidence for this is suggested by the parallel increase in Na+-K+ATPdse in the adapted tissues. For example, the 43% increase in membrane surface density seen in the outer medullary collecting tubule in potassium adaptation roughly corresponds to the 35% increase in Na+K+-ATPase activity of the outer medullary portion of the kidney (17). In the colon, although the 2-fold increase in enzyme activity which has been observed (6) is not quite matched by the increase in membrane surface density, the direction of change is the same and the difference may be accounted for by a heterogeneity of the tissue utilized for enzyme analysis. A parallel appears to be present in nonmammalian tissues. The euryhaline teleost Gristcwsfws t r c w l m f n demonstrates striking changes in the basolateral membrane of the renal tubule during adaptation from fresh
158
MICHAEL KASHGARIAN
FIG.7. Electron micrograph of basolateral interdigitations of outer medullary collecting duct of a potassium-adapted rat. The complexity of the lateral membranes is markedly increased. This is reflected in the increase in S, determined stereologically. x 12,500.
water to sea water (21). A WO increase in basolateral membrane surface density has been observed and this appears to correspond to the increase in ion absorption as reflected by a lowering of the urine osmolarity during such adaptation. Although no enzyme analyses have been carried out in this particular species of fish, in other euryhaline teleosts such as the killifish, adaptation to fresh water is accompanied by an increase in Na+K+-ATPase of renal tissue (12). Similar qualitative structural changes have also been observed in the opposite direction in the renal tubule of the rainbow trout, Snlrno griirdneri, when such fish are adapted from fresh water to salt water ( / I ) .The nasal salt glands of aquatic birds also appear to undergo similar changes in both enzymatic activity and membrane structure during changes of salt intake ( 9 ) . It should also be noted that such an increase in the basolateral membrane surface area is accompanied by an increase in the length and complexity of the intercellular spaces. The importance of the paracellular pathway for transepithelial fluid flow is discussed in other articles in this volume. The structural changes observed here could alter intercellular osmotic gradients o r the paracellular conductance which could in turn have major effects on net transepithelial movement of ions and fluid. It
MEMBRANE SURFACE CHANGES IN ION TRANSPORT
159
is thus apparent that the structural changes associated with ionic adaptation could affect transepithelial ionic movement either as a result of changes in some active component such as in transport enzyme sites o r through alterations of passively induced osmotic fluid flow through the intercellular channels. These observations lead to yet another area of speculation in relation to the mechanism of induction of these membrane changes. In the studies described here, membrane changes were associated with changes in potassium intake, glucocorticoid levels, and mineralocorticoid levels. I t would be interesting if a single effector mechanism could account for all of these environmental changes. Studies in teleosts have suggested that the anterior pituitary hormone, prolactin, appears to be the effector hormone responsible for control of adaptation to fresh water. Its importance has been demonstrated in the killifish. F/rndrr/ir.shc~to.oc/itri.c (/,?), and several species of salmon and trout ( 2 , 8). Furthermore, in studies of the stickleback fish, the structural changes of ionic adaptation have been accelerated as well as duplicated by the action of prolactin (22). Since in humans sodium depletion and steroid therapy are associated with an increase in serum prolactin, it is interesting to speculate that this hormone may indeed be a common mediator involved in the structural and functional adaptations described here. REFERENCES I . Bastl, C. P., Hayslett. J . P., and Binder, H. J . (1977). Role of glucocorticoids on ion transport and transmural potential difference in colon. Cliii. Reg. 25, 306. 2. Bentley. P. J. ( 1971). "Endocrines and Osmoregulation." Springer-Verlag, Berlin and
New York. 3 . Berliner. R. W . , Kennedy, T. G . , and Hilton. J . 0.(1950). Renal mechanisms for excretion of potassium. A m . J. Physiol. 162, 348-367. 4. Ensor. D. M., and Ball, J. N. (1972). Prolactin and osmoregulation. Fed. Pro(,. Fed. A i i i . Soc. E.vp. B i d . 31, 1615-1623. 5 . Finkelstein, F. 0..and Hayslett, J . P. (1975). Role of medullary Na-K ATPase in renal l . S74-528. potassium adaptation. A m . J. P h y ~ i ~229, 6. Fisher, K., Binder, H . J . , and Hayslett. J . P. (1976). Potassium secretion by colonic mucosal cells after potassium adaptation. A m . J . Physiol. 231, 987-994. 7. Hayes, C. P., McLeod, M . E., and Robinson, R. R . (1967). An extrarenal mechanism for the maintenance of potassium balance in severe chronic renal failure. 7'nrri.s. Assoc. A m . Physician.c 80, 207-216. 8 . Hoar, W . S . , and Randall, D. J . (1969). "Fish Physiology." Academic Press, New York. 9. Holmes, W . N. (1972). Regulation of electrolyte balance in marine birds. Fed. Proc. Fod. A m . Soc. E x p . B i d . , 31, 1587-1598. 10. Jorgensen, P. L. (1969). Regulation of Na-K ATPase in the rat kidney. 11. The effect of adolsterone on the activity in kidneys of adrenalectomized rat. Biochini. Biophy.~. Acrcc 192, 326-334.
160
MICHAEL KASHGARIAN
II, Kashgarian, M . (1977). Unpublished observations. 12. Pickford, G. E., Griffith, R. W., Torretti, J., Hendler, E., and Epstein, F. H. (1970).
13. 14. 15.
16. 17.
18. 19. 20.
21. 22.
Branchial reduction and renal stimulation of Na-K ATPase by prolactin in hypophysectomized killifish in fresh water. Nuture (London) 228, 378. Rastegar, A., Hayslett, J . P., and Kashgarian, M . (1980). Changes in membrane surfaces of collecting duct cells in potassium adaptation. Kidney I n r . (in press). Schon, D. A., and Hayslett, J . P. (1978). Potassium adaptation in medullary collecting duct. Kidney I n r . 14, 780. Schon, D . A., Silva, P., and Hayslett, J. P. (1974). The mechanism of renal potassium excretion in uremia. A m . J . Physiol. 227, 1323-1330. Silva, P., Charney, A. N., and Epstein, F. H. (1975). Potassium adaptation and Na-K ATPase activity in mucosa of colon. A m . J . Physiol. 22Y, 1576-1579. Silva, P., Hayslett, J . P., and Epstein, F. H. (1973). The role of Na-K ATPase in potassium adaptation. J . C ' h . lnves/. 52, 2665-267 I . Taylor, C. R., Hayslett, J . P., and Kashgarian, M. (1978). Structural adaptation of cell membrane surfaces to compensatory increases in potassium secretion. Proc. lnr. C'ongr. Neplzrol., 7/11 C9. Thatcher, J . S . , and Radike, A. W. (1947). Tolerance to potassium intoxication in the albino rat. Am. J . Physiol. 151, 138-146. Weibel, E. R. ( 1969). Stereological principles for morphometry in electron microscopic cytology. I n ! . R e v . Cytol. 26, 335. Wendelaar Bonga, S. E., and Veenhuis, M . (1974). The membranes of basal labyrinth in kidney cells in the anandromous teleost Gusterosreus nculearu in adaptation from sea water to fresh water. J . Cell Sci. 14, 587-609. Wendelaar Bonga, S . E. ( 1976). The effect of prolactin on kidney structure of euryhaline teleost Gusterosteus uculrtrrci during adaptation to fresh water. Cell 7issue Rev. 166, 3 19-338.