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Salt and water transport by epithelial cells
Medical Intelligence Am J Otolaryngol 3:361-366, 1982
Salt and Water Transport by
Epithelial Cells KENNETHR. SPRING, D.M.D., PH.D, Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cellular swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cellular shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described and a number of experiments are discussed in which cellular volume determinations provided significant new information about the dynamic behavior of epithelia. (Key words: Epithelial cell volume; Solute transport.)
quantitated. We developed an optical microscopic method for the repetitive determination of epithelial cell volume, The technique for the measurement of the size and shape of epithelial cells is described and various applications of cellular volume measurements are considered,
Epithelial cells function to transport fluid from one surface to the other; this transepithelia] transport is dependent on the metabolism of the cells. The transport of a solute such as NaC1 results in net fluid flow because the hydraulic conductivity of the epithelial cell is sufficiently great to preclude any substantial transepithelial gradient in osmotic pressure. Therefore, trar, sport of solute results in proportionate movement of water and the mucosal and serosaI bathing solutions remain virtually equal in osmolality. The transport of large quantities of solute and water by epithelial cells necessitates substantial transcellular flows of these substances. Many investigators have concluded that the epithelial cell could not possibly withstand such rapid flows of salts and water. T h e y propose that transepithelial fluid m o v e m e n t s are accomplished by an extracellular (paracellular] route, 1-7 In this report I review evidence favoring a transcellular route for transported fluid in Necturus gallbladder epithelial cells. Study of the significance of transcellular flows in the transepithelial movement of fluid requires a method for the rapid measurement of cell volume, so that water flow into or out of the cell may be
MEASUREMENT OF EPITHELIAL CELL VOLUME
Quantitative determinations of epithelial cell volume have been made from electron micrographs of serial sections; s'9 however, studies of the dynamic behavior of the cells were not possible by this technique because of the n e e d for fixation and staining. When Necturus gallbladders or other epithelia are mounted in a thin perfusion chamber, the cell outlines m a y be readily visualized with the light microscope. I~ The use of microscopic optics with an extremely shallow depth of field permits t h e " optical sectioning" of a cell, so that only a thin cross section of the cell is in focus at one time; the remainder of the cell above and below the focal plane is invisible. Examples of such optical sections of a Necturus gallbladder epithelial cell are shown in Figure 1. The use of high magnification (I00• differential interference contrast optics reduces the optical section thickness to 0.5 ~ m or less. The cells are visualized during an experiment by the use of a low-light-level television camera, and the video images of the optical sections are stored on a video disk. The size and shape of a cell may be adequately defined by eight to ten optical sections from the level of the apical surface to the
Received April 23, 1982. Accepted for publication May 24, 1982. Presented at the Midwinter Research Meeting of the Association for Research in Otolaryngology, St. Petersburg Beach, Florida, January 18-21, 1982. * Laboratory of Kidney and Electrolyte Metabolism, Building 10, Room 6N307, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205. Address correspondence and reprint requests to Dr.
Spring.
361
EPITHELIALCELLSOLUTETRANSPORT level of the underlying basement membrane. The microscope focus and video recording are both under computer control. A series of records of the sections through a cell requires about I to 1.5 seconds, and a complete series of sections can be repeated every 6 seconds. ~2'16At a later time the video images are replayed, and the cell outlines are traced electronically to determine the crosssectional area and perimeter of each optical section. Cellular volume is calculated from the area Of each section and the k n o w n displacements of focus between sections. 12"~7This m e t h o d enables the frequent determination of the volume of an epithelial cell during the course of a change in the composition of either bathing solution. Inasmuch as the only parameter measured is the volume of ah epithelial cell, care m u s t be taken to design experiments that produce interpretable effects on that parameter.
HYDRAULIC CONDUCTIVITY (Lp) OF EPITHELIAL CELLS Although there has been considerable debate over the magnitude of epithelial permeability to water, 4"~s'~9little effort has been directed towards m e a s u r e m e n t of the permeability to water of epithelial cell m e m b r a n e s . R e c e n t l y the hydraulic c o n d u c t i v i t y of Necturus gallbladder epithelial cells was measured by determining the rate of change of cellular volume in response to a change in the osmolality of either bathing solution. 12"15'16The hydraulic conductivity of the cell membranes was very high (0.055 to 0.12 crrdsec), and only small transmembrane osmotic gradients (about 3.5 mOsm) would be required to accomplish normal rates of fluid absorption. Since the directional transport of fluid requires favorable gradients in water activity as well as adequate hydraulic conductivity, there must be differences in osmolality between the cell interior and the solutions bathing the cell surfaces. The entry of fluid into the gallbladder cell across the apical membrane must be the consequence of a lower water activity within the cell than in the mucosal medium. We calculated from our Lp and f l u i d absorption data that the osmolality of the epithelial cell must exceed that of the luminal fluid by approximately 2.4 m O s m to achieve a normal rate of fluid absorption. ~2"~5The exit of
American Journal of QtolaryngoJogy 362
water from the cell must similarly be the result of an osmotic gradient across the basolateral membrane. We calculate from the Lp of the basolateral membrane that the cell must be bathed on its basolateral surface by a solution approximately 1.1 mOsm hypertonic to its interior or 3.5 m O s m hyperosmotic to the mucosal bathing solution. The a b s o r b a t e w o u l d t h e n be 3.5 m O s m hyperosmotic to the bathing solution, a difference of 1.8 per cent from iso-osmolality. A deviation in osmolality of this m a g n i t u d e is w i t h i n the measurement error of current m e t h o d s for osmolality determination and could not be detected with certainty. Similar conclusions have been reached previously on the basis of mathematical models of epithelial fluid transport. ~'2~ The high Lp of the cell membrane removes the need for consideration of standing osmotic gradients ls'2~ or other more exotic m e c h a n i s m s 't for iso-osmotic fluid absorption. The required osmotic gradients are miniscule and the intercellular space solute concentration profile a n d interspace g e o m e t r y are not s i g n i f i c a n t factors during normal fluid absorption. 6,.2,23 A further consequence of the ready m o v e m e n t of water across epithelial cell membrane is that solute entry into or exit from an epithelial cell is followed by water movement in about the same ratio of solute/solvent as in the b u l k solution. The high cell membrane Lp reported for the Necturus gallbladder epithelium virtually eliminates the possibility of significant paracellular fluid flow in that tissue. 12 The driving force for fluid movement across the tight junctions is the difference in osmotic pressure between the basolateral interstitium and the muscosal bathing solution. The osmotic pressure difference across the tight junction is the same as that across the entire cell; therefore, equal fluid flows across the two pathways (junctional and cellular) w o u l d require equal h y d r a u l i c c o n d u c tivities. The available area of the tight junction is 10 -4 times that of the epithelial cell apical membrane; therefore, the junctional Lp m u s t be about 10 -4 times that of the apical cell membrane. Significant transjnnctiona] water f l o w w o u l d require water p e r m e a b i l i t y v a l u e s of the t i g h t junction in the range of 5 meters/sec, a physically unrealistic value. Cell m e m b r a n e water permeability is sufficiently high that all trans-
Figure I (facingpage). Optical sectioning is illustrated by light-microscopic (differentialinterference contrast) images of Necturus gallbladderepithelium. Each image was recorded after the focus was adjusted 3/~mtoward the base of the cell. Image 1 is approximatelyat the level af the tight junctions between the ceils. Image2 is 3/~m serosal to the tight junction, and each subsequent image is 3 ~m closer to the serosal side. Notice that intercellularbridges are most prominent in frames 2 through 5 (3/~m to 12 p.m beneath the tight junctions). Cell boundaries are indistinct in frame 8 and strands of connective tissue are visible. Magnification630• Photographs by Harry G. Schaefer. (Reprinted from Spring and Persson,TM with permission of Raven Press,)
SPRING
Volume 3 Number 5 September 1982
363
EPITHELIAL CELL SOLUTE TRANSPORT
ported fluid takes a transcellular route, driven by small osmotic gradients. TRANSCELLULAR NaCI TRANSPORT
American Journal
of Otolaryngalogy 364
Necturus gallbladder epithelial cells transport NaC1 from their apical to their basolateral surfaces. The bile facing the apical surface is thereby concentrated by the removal of NaC1 and water. The mechanism of NaC1 transport by these cells has been studied by several groups of investigators. 1~ In most absorptive epithelia the rate-limiting step for the transcellular transport of Na + or NaC1 lies at the apical membrane of the cell. ~4"25"82-3~Sodium entry has been shown to be a saturable function of the Na + concentration in the mucosal bathing solution, suggesting that the entry step could be carrier-mediated. There is a consensus that NaC1 enters Necturus gallbladder cells across the apical membrane, driven by the Na + concentration gradient across the membrane. 1~ Electroneutral, mediated entry of NaC1 into epithelial cells across the apical membrane has been reported to occur in a number of low-resistance epithelia. 'S'~'l-4~Indeed, this phenomenon may be a general mechanism for the entry of salt into epithelial cells, inasmuch as most epithelial cells have chloride activities far above the equilibrium value2 A high intracellular chloride activity is the consequence of the coupled nature of the entry step for NaC1 into the epithelial cell. The intracellular chloride activity is maintained above equilibrium by the continuing entry of NaC1 across the apical membrane as a result of the favorable electrochemical gradient for No+.3,2~,35 This entry of NaC1 maintains the cellular osmolality above that of the apical bathing solution. Normally the entry of NaC1 across the apical membrane is exactly balanced by its exit across the basolateral cell membrane; the exit is due to active Na + transport by the epithelial cell. The relationship between the rate of Na + entry into epithelial cells and the rate of Na + exit has been the subject of considerable research. 32,42,43 A negative-feedback hypothesis has been put forth which suggests that increased Na + entry elevates intracellular Na + concentration and that this elevated Na + concentration results in a reduction of Na + entry and the restoration of the original intracellular Na + concentration. 41,43 The high hydraulic conductivity of the cell membranes of leaky epithelia leads to the flow of water into or out of the cell in response to a change in the solute content of the cell. ~2 The
maintenance of a stable volume by an epithelial cell would seem to be a consequence of the balance between solute entry and exit across the two faces of the cell. Indeed, this has been demonstrated in a number of different ways in Necturus gallbladder epithelial cells, 1~ as well as in isolated rabbit proximal tubule segmentsJ 4 Because two processes compete with one another for the solute content of the cell, the volume of the cell may be predictably altered by cessation of solute entry or exit. For example: (11 removal of mucosal Na + or C1- stops entry of both solutes and abolishes fluid absorption by Necturus gallbladder epithelium. 1~ Cessation of NaC1 entry leads to cell shrinkage because NaC1 exit continues as long as the intracellular Na concentration is sufficiently high to maintain the Na+,K+-ATPase activity. Cellular volume predictably decreases under these circumstances as the intracellular NaC1 pool is depleted and the solute content of the cell decreases. ~7 The magnitude and rate of this decrease in cell volume have been used to estimate the NaCI transport pool in the cell, as well as the rate of NaCI exit from the cell due to active Na + transport. ~ (2) NaC1 exit may be blocked by the inhibitor ouabain. 1~ A d d i t i o n of 10 -4 M o u a b a i n to the serosal bathing solution of Necturus gallbladder results in cellular swelling because NaC1 exit has been blocked but entry continues. The rate of cellular s w e l l i n g and the m a g n i t u d e of the m a x i m u m volume increase are a function of the ionic composition of the apical bathing solution. The NaC1 entry step in Necturus gallbladder epithelium may be characterized by analysis of the rate of cellular swelling in the presence of ouabain. 1~ The kinetic characteristics and inhibitor sensitivity of this process are consistent with the coupled, neutral transport of NaC1. In summary, fluid absorption by Necturus gallbladder (and possibly other leaky epithelia) may be explained as follows: 1. NaC1 enters the epithelial cell across the apical membrane by a carrier-mediated process in which the co-transport of chloride is driven by the gradient for sodium, This entry process is the p r i n c i p a l m o d e of NaC1 m o v e m e n t associated with transepithelial transport and is rate-limiting for transepithelial fluid transport. 2. The entry of NaC1 into the cell maintains the cellular osmolality slightly higher than that of the mucosal bath (about 2 mOsm). Water flows from the mucosal bathing solution into the cell, driven by the gradient in activity from mucosal bath to cell.
SPRING 3. S o d i u m is t r a n s p o r t e d o u t of t h e c e l l a c r o s s the basolateral membrane b y t h e N a + , K +A T P a s e . T h e m o d e of c h l o r i d e e x i t f r o m t h e cell is u n c e r t a i n . 2s'4s'4s T h e t r a n s p o r t o f NaG1 i n t o t h e b a s o l a t e r a l i n t e r s t i t i a l s p a c e i n c r e a s e s its o s m o l a l i t y b y a b o u t 1 m O s m a b o v e t h a t of the cell. Water moves from cell to basolateral int e r s t i t i u m , d r i v e n b y t h e g r a d i e n t i n its a c t i v i t y . 4. A s m a l l h y d r o s t a t i c p r e s s u r e ( a b o u t 3 c m H2014 d e v e l o p s i n t h e b a s o l a t e r a l i n t e r c e l l u l a r space and drives the fluid across the submucosal connective tissue. T r a n s e p i t h e l i a l f l u i d f l o w is t r a n s c e l l u l a r , with fluid entering the cell across the apical membrane and exiting across the basolateral membrane. The lateral intercellular spaces constitute the final common pathway for transp o r t e d f l u i d b u t do n o t p l a y a n i m p o r t a n t r o l e in s o l u t e - s o l v e n t c o u p l i n g . A s p e c i f i c r o l e for t h e l e a k y t i g h t j u n c t i o n is n o t r e a d i l y a p p a r e n t at this time, References 1. DiBona DR, Civan MM: Pathways for movement of ions and water across toad urinary bladder: I, Anatomic site of transepithelial shunt pathways. I Membrane Biol 12:101-128, 1973 2. DiBona DR, Civan MM, Leaf A: The anatomic site of the transepithelial permeability barriers of toad bladder. J Cell Biol 40:1-7, 1969 3. Frizzell RA, Duffy ME: Chloride activities in epithelia. Fed Proc 39:2860-2864, 1980 4. Hill A: Salt-water coupling in leaky epithelia. J Membrane Biol 55:117-182, 1980 5. Hill AE, Hill BS: Sucrose fluxes and junctional water flow across Necturus gallbladder epithelium. Proc R Soc Lend B 200:163-174, 1978 6. Sackin H, Boulpaep EL: Models for coupling of salt and water transport. ] Gen Physiol 66:671-733, 1975 7. Schafer JA, Patlak CS, Troutman SL, etah Volume absorption in the pars recta. II. Hydraulic conductivity coefficient. Am J Physiol (Renal Fluid Electrolyte Physiol 3) 234:F340-F348, 1978 8. Blom H, Helander HD: Quantitative electron microscopical studies on in vitro incubated rabbit gallbladder epithelium. J Membrane Biol 37:45-61, 1977 9. Welling DJ, Welling LW: Cell shape as an indicator of volume reabsorption in proximal nephron. Fed Proc 38:121-127, 1979 10. Ericson A-C, Spring KR: Coupled NaC1 entry into Necturus gallbladder epithelial cells. Am J PhysiohCell (in press) 11. Ericson H-C, Spring KR: Volume regulation by Necturus gallbladder: hypertonicity activates apical Na-H and C1-HCO, exchange. Am J Physiol:Cell (in press} 12. Persson B-E, Spring KR: Gallbladder epithelial cell hydraulic water permeability and volume regulation, ] Gen Physiol 79:481-505, 1982 13, Spring KR: Optical techniques for the evaluation of epithelial transport processes. Am J Physiol 237 (Renal Fluid Electrolyte Physiol 6):F167-F174, 1979 14. Spring KR, Hope A: Size and shape of the lateral intercellular spaces in a living epithelium, Science 200:54-58, 1978
15. Spring KR, Hope A, Persson B-E: Quantitative light microscope studies of epithelial fluid transport, in: Ussing HH, Bindslev N, Lessen NA, et al [eds,): Water Transport Across Epithelia. Munksgaard, Copenhagen, 1981. pp 190-205 16. Spring KR, Persson B-E: Quantitative light microscopy and epithelial function, in: Macknight ADC, Leader ]P (eds.]; Epithelial Ion and Water Transport. Raven Press, New York, 1961, pp 15-21 17, Spring KR, Hope A: Fluid transport and the dimensions of cells and interspaces of living Necturus gallbladder. ]~Can Physiol 73:287-305, 1979 18. Diamond JM: Osmotic water flow in leaky epithelia. J Membrane Biol 51',195-216, 1979 19. van Os CH, Wiedner G, Wright EM: Volume flows across gallbladder epithelium induced by small hydrostatic and osmotic gradients, J Membrane Biol 49:1-20, 1979 20. Liebovitch LS, Weinbanm S: A model of epithelial water transport. Biophys J 35:315-318, 1981 21, Weinstein AM, Stephenson JL: Electrolyte transport across a simple epithelium, Biophys J 27;165-186, 1979 22. Weinstein AM, Stephanson JL, Spring KR: The coupled transport of water, in: Bonting LK, dePont J (eds.): Membrane Transport. Volume 2, New Comprehensive Biochemistry. Elsevier, Amsterdam, 1981, pp 311-349 23. Weinstein AM, Stephenson JL: Coupled water transport in standing gradient models of the lateral intercellular space. Biophys J 35:167-191, 1981 24. Fromter E: The route of passive ion movement through the epithelium of Necturus gallbladder. J Membrane Biol 8:259-301, 1972 25, Garcia-Diaz JF, Armstrong WMcD: The steady-state relationship between sodium and chloride transmembrans electrochemical potential differences in Necturus gallbladder. J Membrane Biol 55:213-222, 1980 26. Graf J, Giobisch G: Intracellular sodium activity and sodium transport in Nectums gallbladder epithelium, J Membrane Biol 47:327-355, 1979 27. Reuss L, Grady TP: Effects of external sodium and cell membrane potential on intracellular chloride activity in gallbladder epithelium. J Membrane Biol 51:15-31, 1979 28. Reuss L, Weinman SA: Intraeellular ionic activities and intramembrane electrochemical potential differences in gallbladder epithelium. J Membrane Biol 49:345362, 1979 29. Reuss L, Weinman SA, Grady TO: Intracellular K+ activity and its relation to basolateral membrane ion transport in Necturus gallbladder epithelium. J Con Physiol 76:33-52, 1980 39. Rose RC, Nahrwold DL: Electrolyte transport in Necturus gallbladder: the role of rheoganic Na transport. Am J Physiol [Gastroint Liver Physiol 1):G358-G365, 1980 31, van Os CH, Slegers ~FG: The electrical potential profile of gallbladder epithelium. ] Membrane Biol 24:341363, 1975 32. Chase HS, A1-Awqati Q: Regulation of the sodium permeability of the luminal border of toad bladder by intracelluiar sodium and calcium. J Gen Physiol 77:693-712, 1981 33. Cremaschi D, Henin S: Na+ and CI- transepithelial routes in rabbit gallbladder. Pfluegers Arch 361:33-41, 1975 34. Frizzell R.A, Dugas MC, Schultz SG: Sodium chloride transport by rabbit gallbladder. J Gen Physiol 65:769-795, 1975 35. Frizzell RA, Field M, Schultz SG: Sodium-coupled chloride transport by epithelial tissues. Am J Physiol 236 (Renal Fluid Electrolyte Physiol 5):F1-Fg, 1979 36. Kimura C, Spring K.R:Luminal Na+ entry into Necturus
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EPITHELIAL CELL SOLUTE TRANSPORT
37. 38. 39. 40.
41.
American Journal af Otolaryngology 366
proximal tubule cells. Am J Physiol 236 (Renal Fluid Electrolyte Physiol 5):F295-F301, 1979 Spring KR, Giebisch G: Kinetics of Na + transport in Necturus proximal tubule. J Gen Physiol 70:307-328, 1977 Spring KR, Kimura G: Intracellular ion activities in Necturus proximal tubule. Fed Proc 38:2729-2732, 1979 Henin S, Cremaschi D: Transcellular ion route in rabbit gallbladder. Pfluegers Arch 355:125-139, 1975 Silva P, Staff F, Field M, et ah Mechanism of active chloride secretion in shark rectal gland: role of Na-KATPase in chloride transport. Am J Physioi 233 (Renal Fluid Electrolyte Physiol 2):F298-F306, 1977 Spring KR, Kimura G: Chloride reabsorpfion by rena] proximal tubules of Necturus. J Membrane Bial 38:233-254, 1978
42. Lewis SA: A reinvestigation of the function of the mammalian urinary bladder. Am J Physiol 232 (Renal Fluid Electrolyte Physiol 1):F187-F195, 1977 43. Taylor A, Windhager EE: Possible role of cytosolic calcium and Na-Ca exchange in regulation of transepithelial sodium transport, Am J Physiol 236 (Renal Fluid Electrolyte Physiol 5):F505-F512, 1979 44. Linshaw M: Effect of metabolic inhibitors on renal tubule cell volume. Am J Physiol 239 (Renal Fluid Electrolyte Physiol 8):F571-F577, 1980 45. Reuss L: Electrical properties of the cellular transepithelial pathway in Necturus gallbladder: III. Ionic permeability of the basolateral cell membrane. J Membrane Biol 47:239-259, 1979 46. Shindo T, Spring IGR: Chloride movement across the basolateral membrane of proximal tubule cells. J Membrane Biol 58:35-42, 1981
Salt and Water Transport by
Epithelial Cells KENNETHR. SPRING, D.M.D., PH.D, Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cellular swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cellular shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described and a number of experiments are discussed in which cellular volume determinations provided significant new information about the dynamic behavior of epithelia. (Key words: Epithelial cell volume; Solute transport.)
quantitated. We developed an optical microscopic method for the repetitive determination of epithelial cell volume, The technique for the measurement of the size and shape of epithelial cells is described and various applications of cellular volume measurements are considered,
Epithelial cells function to transport fluid from one surface to the other; this transepithelia] transport is dependent on the metabolism of the cells. The transport of a solute such as NaC1 results in net fluid flow because the hydraulic conductivity of the epithelial cell is sufficiently great to preclude any substantial transepithelial gradient in osmotic pressure. Therefore, trar, sport of solute results in proportionate movement of water and the mucosal and serosaI bathing solutions remain virtually equal in osmolality. The transport of large quantities of solute and water by epithelial cells necessitates substantial transcellular flows of these substances. Many investigators have concluded that the epithelial cell could not possibly withstand such rapid flows of salts and water. T h e y propose that transepithelial fluid m o v e m e n t s are accomplished by an extracellular (paracellular] route, 1-7 In this report I review evidence favoring a transcellular route for transported fluid in Necturus gallbladder epithelial cells. Study of the significance of transcellular flows in the transepithelial movement of fluid requires a method for the rapid measurement of cell volume, so that water flow into or out of the cell may be
MEASUREMENT OF EPITHELIAL CELL VOLUME
Quantitative determinations of epithelial cell volume have been made from electron micrographs of serial sections; s'9 however, studies of the dynamic behavior of the cells were not possible by this technique because of the n e e d for fixation and staining. When Necturus gallbladders or other epithelia are mounted in a thin perfusion chamber, the cell outlines m a y be readily visualized with the light microscope. I~ The use of microscopic optics with an extremely shallow depth of field permits t h e " optical sectioning" of a cell, so that only a thin cross section of the cell is in focus at one time; the remainder of the cell above and below the focal plane is invisible. Examples of such optical sections of a Necturus gallbladder epithelial cell are shown in Figure 1. The use of high magnification (I00• differential interference contrast optics reduces the optical section thickness to 0.5 ~ m or less. The cells are visualized during an experiment by the use of a low-light-level television camera, and the video images of the optical sections are stored on a video disk. The size and shape of a cell may be adequately defined by eight to ten optical sections from the level of the apical surface to the
Received April 23, 1982. Accepted for publication May 24, 1982. Presented at the Midwinter Research Meeting of the Association for Research in Otolaryngology, St. Petersburg Beach, Florida, January 18-21, 1982. * Laboratory of Kidney and Electrolyte Metabolism, Building 10, Room 6N307, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205. Address correspondence and reprint requests to Dr.
Spring.
361
EPITHELIALCELLSOLUTETRANSPORT level of the underlying basement membrane. The microscope focus and video recording are both under computer control. A series of records of the sections through a cell requires about I to 1.5 seconds, and a complete series of sections can be repeated every 6 seconds. ~2'16At a later time the video images are replayed, and the cell outlines are traced electronically to determine the crosssectional area and perimeter of each optical section. Cellular volume is calculated from the area Of each section and the k n o w n displacements of focus between sections. 12"~7This m e t h o d enables the frequent determination of the volume of an epithelial cell during the course of a change in the composition of either bathing solution. Inasmuch as the only parameter measured is the volume of ah epithelial cell, care m u s t be taken to design experiments that produce interpretable effects on that parameter.
HYDRAULIC CONDUCTIVITY (Lp) OF EPITHELIAL CELLS Although there has been considerable debate over the magnitude of epithelial permeability to water, 4"~s'~9little effort has been directed towards m e a s u r e m e n t of the permeability to water of epithelial cell m e m b r a n e s . R e c e n t l y the hydraulic c o n d u c t i v i t y of Necturus gallbladder epithelial cells was measured by determining the rate of change of cellular volume in response to a change in the osmolality of either bathing solution. 12"15'16The hydraulic conductivity of the cell membranes was very high (0.055 to 0.12 crrdsec), and only small transmembrane osmotic gradients (about 3.5 mOsm) would be required to accomplish normal rates of fluid absorption. Since the directional transport of fluid requires favorable gradients in water activity as well as adequate hydraulic conductivity, there must be differences in osmolality between the cell interior and the solutions bathing the cell surfaces. The entry of fluid into the gallbladder cell across the apical membrane must be the consequence of a lower water activity within the cell than in the mucosal medium. We calculated from our Lp and f l u i d absorption data that the osmolality of the epithelial cell must exceed that of the luminal fluid by approximately 2.4 m O s m to achieve a normal rate of fluid absorption. ~2"~5The exit of
American Journal of QtolaryngoJogy 362
water from the cell must similarly be the result of an osmotic gradient across the basolateral membrane. We calculate from the Lp of the basolateral membrane that the cell must be bathed on its basolateral surface by a solution approximately 1.1 mOsm hypertonic to its interior or 3.5 m O s m hyperosmotic to the mucosal bathing solution. The a b s o r b a t e w o u l d t h e n be 3.5 m O s m hyperosmotic to the bathing solution, a difference of 1.8 per cent from iso-osmolality. A deviation in osmolality of this m a g n i t u d e is w i t h i n the measurement error of current m e t h o d s for osmolality determination and could not be detected with certainty. Similar conclusions have been reached previously on the basis of mathematical models of epithelial fluid transport. ~'2~ The high Lp of the cell membrane removes the need for consideration of standing osmotic gradients ls'2~ or other more exotic m e c h a n i s m s 't for iso-osmotic fluid absorption. The required osmotic gradients are miniscule and the intercellular space solute concentration profile a n d interspace g e o m e t r y are not s i g n i f i c a n t factors during normal fluid absorption. 6,.2,23 A further consequence of the ready m o v e m e n t of water across epithelial cell membrane is that solute entry into or exit from an epithelial cell is followed by water movement in about the same ratio of solute/solvent as in the b u l k solution. The high cell membrane Lp reported for the Necturus gallbladder epithelium virtually eliminates the possibility of significant paracellular fluid flow in that tissue. 12 The driving force for fluid movement across the tight junctions is the difference in osmotic pressure between the basolateral interstitium and the muscosal bathing solution. The osmotic pressure difference across the tight junction is the same as that across the entire cell; therefore, equal fluid flows across the two pathways (junctional and cellular) w o u l d require equal h y d r a u l i c c o n d u c tivities. The available area of the tight junction is 10 -4 times that of the epithelial cell apical membrane; therefore, the junctional Lp m u s t be about 10 -4 times that of the apical cell membrane. Significant transjnnctiona] water f l o w w o u l d require water p e r m e a b i l i t y v a l u e s of the t i g h t junction in the range of 5 meters/sec, a physically unrealistic value. Cell m e m b r a n e water permeability is sufficiently high that all trans-
Figure I (facingpage). Optical sectioning is illustrated by light-microscopic (differentialinterference contrast) images of Necturus gallbladderepithelium. Each image was recorded after the focus was adjusted 3/~mtoward the base of the cell. Image 1 is approximatelyat the level af the tight junctions between the ceils. Image2 is 3/~m serosal to the tight junction, and each subsequent image is 3 ~m closer to the serosal side. Notice that intercellularbridges are most prominent in frames 2 through 5 (3/~m to 12 p.m beneath the tight junctions). Cell boundaries are indistinct in frame 8 and strands of connective tissue are visible. Magnification630• Photographs by Harry G. Schaefer. (Reprinted from Spring and Persson,TM with permission of Raven Press,)
SPRING
Volume 3 Number 5 September 1982
363
EPITHELIAL CELL SOLUTE TRANSPORT
ported fluid takes a transcellular route, driven by small osmotic gradients. TRANSCELLULAR NaCI TRANSPORT
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of Otolaryngalogy 364
Necturus gallbladder epithelial cells transport NaC1 from their apical to their basolateral surfaces. The bile facing the apical surface is thereby concentrated by the removal of NaC1 and water. The mechanism of NaC1 transport by these cells has been studied by several groups of investigators. 1~ In most absorptive epithelia the rate-limiting step for the transcellular transport of Na + or NaC1 lies at the apical membrane of the cell. ~4"25"82-3~Sodium entry has been shown to be a saturable function of the Na + concentration in the mucosal bathing solution, suggesting that the entry step could be carrier-mediated. There is a consensus that NaC1 enters Necturus gallbladder cells across the apical membrane, driven by the Na + concentration gradient across the membrane. 1~ Electroneutral, mediated entry of NaC1 into epithelial cells across the apical membrane has been reported to occur in a number of low-resistance epithelia. 'S'~'l-4~Indeed, this phenomenon may be a general mechanism for the entry of salt into epithelial cells, inasmuch as most epithelial cells have chloride activities far above the equilibrium value2 A high intracellular chloride activity is the consequence of the coupled nature of the entry step for NaC1 into the epithelial cell. The intracellular chloride activity is maintained above equilibrium by the continuing entry of NaC1 across the apical membrane as a result of the favorable electrochemical gradient for No+.3,2~,35 This entry of NaC1 maintains the cellular osmolality above that of the apical bathing solution. Normally the entry of NaC1 across the apical membrane is exactly balanced by its exit across the basolateral cell membrane; the exit is due to active Na + transport by the epithelial cell. The relationship between the rate of Na + entry into epithelial cells and the rate of Na + exit has been the subject of considerable research. 32,42,43 A negative-feedback hypothesis has been put forth which suggests that increased Na + entry elevates intracellular Na + concentration and that this elevated Na + concentration results in a reduction of Na + entry and the restoration of the original intracellular Na + concentration. 41,43 The high hydraulic conductivity of the cell membranes of leaky epithelia leads to the flow of water into or out of the cell in response to a change in the solute content of the cell. ~2 The
maintenance of a stable volume by an epithelial cell would seem to be a consequence of the balance between solute entry and exit across the two faces of the cell. Indeed, this has been demonstrated in a number of different ways in Necturus gallbladder epithelial cells, 1~ as well as in isolated rabbit proximal tubule segmentsJ 4 Because two processes compete with one another for the solute content of the cell, the volume of the cell may be predictably altered by cessation of solute entry or exit. For example: (11 removal of mucosal Na + or C1- stops entry of both solutes and abolishes fluid absorption by Necturus gallbladder epithelium. 1~ Cessation of NaC1 entry leads to cell shrinkage because NaC1 exit continues as long as the intracellular Na concentration is sufficiently high to maintain the Na+,K+-ATPase activity. Cellular volume predictably decreases under these circumstances as the intracellular NaC1 pool is depleted and the solute content of the cell decreases. ~7 The magnitude and rate of this decrease in cell volume have been used to estimate the NaCI transport pool in the cell, as well as the rate of NaCI exit from the cell due to active Na + transport. ~ (2) NaC1 exit may be blocked by the inhibitor ouabain. 1~ A d d i t i o n of 10 -4 M o u a b a i n to the serosal bathing solution of Necturus gallbladder results in cellular swelling because NaC1 exit has been blocked but entry continues. The rate of cellular s w e l l i n g and the m a g n i t u d e of the m a x i m u m volume increase are a function of the ionic composition of the apical bathing solution. The NaC1 entry step in Necturus gallbladder epithelium may be characterized by analysis of the rate of cellular swelling in the presence of ouabain. 1~ The kinetic characteristics and inhibitor sensitivity of this process are consistent with the coupled, neutral transport of NaC1. In summary, fluid absorption by Necturus gallbladder (and possibly other leaky epithelia) may be explained as follows: 1. NaC1 enters the epithelial cell across the apical membrane by a carrier-mediated process in which the co-transport of chloride is driven by the gradient for sodium, This entry process is the p r i n c i p a l m o d e of NaC1 m o v e m e n t associated with transepithelial transport and is rate-limiting for transepithelial fluid transport. 2. The entry of NaC1 into the cell maintains the cellular osmolality slightly higher than that of the mucosal bath (about 2 mOsm). Water flows from the mucosal bathing solution into the cell, driven by the gradient in activity from mucosal bath to cell.
SPRING 3. S o d i u m is t r a n s p o r t e d o u t of t h e c e l l a c r o s s the basolateral membrane b y t h e N a + , K +A T P a s e . T h e m o d e of c h l o r i d e e x i t f r o m t h e cell is u n c e r t a i n . 2s'4s'4s T h e t r a n s p o r t o f NaG1 i n t o t h e b a s o l a t e r a l i n t e r s t i t i a l s p a c e i n c r e a s e s its o s m o l a l i t y b y a b o u t 1 m O s m a b o v e t h a t of the cell. Water moves from cell to basolateral int e r s t i t i u m , d r i v e n b y t h e g r a d i e n t i n its a c t i v i t y . 4. A s m a l l h y d r o s t a t i c p r e s s u r e ( a b o u t 3 c m H2014 d e v e l o p s i n t h e b a s o l a t e r a l i n t e r c e l l u l a r space and drives the fluid across the submucosal connective tissue. T r a n s e p i t h e l i a l f l u i d f l o w is t r a n s c e l l u l a r , with fluid entering the cell across the apical membrane and exiting across the basolateral membrane. The lateral intercellular spaces constitute the final common pathway for transp o r t e d f l u i d b u t do n o t p l a y a n i m p o r t a n t r o l e in s o l u t e - s o l v e n t c o u p l i n g . A s p e c i f i c r o l e for t h e l e a k y t i g h t j u n c t i o n is n o t r e a d i l y a p p a r e n t at this time, References 1. DiBona DR, Civan MM: Pathways for movement of ions and water across toad urinary bladder: I, Anatomic site of transepithelial shunt pathways. I Membrane Biol 12:101-128, 1973 2. DiBona DR, Civan MM, Leaf A: The anatomic site of the transepithelial permeability barriers of toad bladder. J Cell Biol 40:1-7, 1969 3. Frizzell RA, Duffy ME: Chloride activities in epithelia. Fed Proc 39:2860-2864, 1980 4. Hill A: Salt-water coupling in leaky epithelia. J Membrane Biol 55:117-182, 1980 5. Hill AE, Hill BS: Sucrose fluxes and junctional water flow across Necturus gallbladder epithelium. Proc R Soc Lend B 200:163-174, 1978 6. Sackin H, Boulpaep EL: Models for coupling of salt and water transport. ] Gen Physiol 66:671-733, 1975 7. Schafer JA, Patlak CS, Troutman SL, etah Volume absorption in the pars recta. II. Hydraulic conductivity coefficient. Am J Physiol (Renal Fluid Electrolyte Physiol 3) 234:F340-F348, 1978 8. Blom H, Helander HD: Quantitative electron microscopical studies on in vitro incubated rabbit gallbladder epithelium. J Membrane Biol 37:45-61, 1977 9. Welling DJ, Welling LW: Cell shape as an indicator of volume reabsorption in proximal nephron. Fed Proc 38:121-127, 1979 10. Ericson A-C, Spring KR: Coupled NaC1 entry into Necturus gallbladder epithelial cells. Am J PhysiohCell (in press) 11. Ericson H-C, Spring KR: Volume regulation by Necturus gallbladder: hypertonicity activates apical Na-H and C1-HCO, exchange. Am J Physiol:Cell (in press} 12. Persson B-E, Spring KR: Gallbladder epithelial cell hydraulic water permeability and volume regulation, ] Gen Physiol 79:481-505, 1982 13, Spring KR: Optical techniques for the evaluation of epithelial transport processes. Am J Physiol 237 (Renal Fluid Electrolyte Physiol 6):F167-F174, 1979 14. Spring KR, Hope A: Size and shape of the lateral intercellular spaces in a living epithelium, Science 200:54-58, 1978
15. Spring KR, Hope A, Persson B-E: Quantitative light microscope studies of epithelial fluid transport, in: Ussing HH, Bindslev N, Lessen NA, et al [eds,): Water Transport Across Epithelia. Munksgaard, Copenhagen, 1981. pp 190-205 16. Spring KR, Persson B-E: Quantitative light microscopy and epithelial function, in: Macknight ADC, Leader ]P (eds.]; Epithelial Ion and Water Transport. Raven Press, New York, 1961, pp 15-21 17, Spring KR, Hope A: Fluid transport and the dimensions of cells and interspaces of living Necturus gallbladder. ]~Can Physiol 73:287-305, 1979 18. Diamond JM: Osmotic water flow in leaky epithelia. J Membrane Biol 51',195-216, 1979 19. van Os CH, Wiedner G, Wright EM: Volume flows across gallbladder epithelium induced by small hydrostatic and osmotic gradients, J Membrane Biol 49:1-20, 1979 20. Liebovitch LS, Weinbanm S: A model of epithelial water transport. Biophys J 35:315-318, 1981 21, Weinstein AM, Stephenson JL: Electrolyte transport across a simple epithelium, Biophys J 27;165-186, 1979 22. Weinstein AM, Stephanson JL, Spring KR: The coupled transport of water, in: Bonting LK, dePont J (eds.): Membrane Transport. Volume 2, New Comprehensive Biochemistry. Elsevier, Amsterdam, 1981, pp 311-349 23. Weinstein AM, Stephenson JL: Coupled water transport in standing gradient models of the lateral intercellular space. Biophys J 35:167-191, 1981 24. Fromter E: The route of passive ion movement through the epithelium of Necturus gallbladder. J Membrane Biol 8:259-301, 1972 25, Garcia-Diaz JF, Armstrong WMcD: The steady-state relationship between sodium and chloride transmembrans electrochemical potential differences in Necturus gallbladder. J Membrane Biol 55:213-222, 1980 26. Graf J, Giobisch G: Intracellular sodium activity and sodium transport in Nectums gallbladder epithelium, J Membrane Biol 47:327-355, 1979 27. Reuss L, Grady TP: Effects of external sodium and cell membrane potential on intracellular chloride activity in gallbladder epithelium. J Membrane Biol 51:15-31, 1979 28. Reuss L, Weinman SA: Intraeellular ionic activities and intramembrane electrochemical potential differences in gallbladder epithelium. J Membrane Biol 49:345362, 1979 29. Reuss L, Weinman SA, Grady TO: Intracellular K+ activity and its relation to basolateral membrane ion transport in Necturus gallbladder epithelium. J Con Physiol 76:33-52, 1980 39. Rose RC, Nahrwold DL: Electrolyte transport in Necturus gallbladder: the role of rheoganic Na transport. Am J Physiol [Gastroint Liver Physiol 1):G358-G365, 1980 31, van Os CH, Slegers ~FG: The electrical potential profile of gallbladder epithelium. ] Membrane Biol 24:341363, 1975 32. Chase HS, A1-Awqati Q: Regulation of the sodium permeability of the luminal border of toad bladder by intracelluiar sodium and calcium. J Gen Physiol 77:693-712, 1981 33. Cremaschi D, Henin S: Na+ and CI- transepithelial routes in rabbit gallbladder. Pfluegers Arch 361:33-41, 1975 34. Frizzell R.A, Dugas MC, Schultz SG: Sodium chloride transport by rabbit gallbladder. J Gen Physiol 65:769-795, 1975 35. Frizzell RA, Field M, Schultz SG: Sodium-coupled chloride transport by epithelial tissues. Am J Physiol 236 (Renal Fluid Electrolyte Physiol 5):F1-Fg, 1979 36. Kimura C, Spring K.R:Luminal Na+ entry into Necturus
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proximal tubule cells. Am J Physiol 236 (Renal Fluid Electrolyte Physiol 5):F295-F301, 1979 Spring KR, Giebisch G: Kinetics of Na + transport in Necturus proximal tubule. J Gen Physiol 70:307-328, 1977 Spring KR, Kimura G: Intracellular ion activities in Necturus proximal tubule. Fed Proc 38:2729-2732, 1979 Henin S, Cremaschi D: Transcellular ion route in rabbit gallbladder. Pfluegers Arch 355:125-139, 1975 Silva P, Staff F, Field M, et ah Mechanism of active chloride secretion in shark rectal gland: role of Na-KATPase in chloride transport. Am J Physioi 233 (Renal Fluid Electrolyte Physiol 2):F298-F306, 1977 Spring KR, Kimura G: Chloride reabsorpfion by rena] proximal tubules of Necturus. J Membrane Bial 38:233-254, 1978
42. Lewis SA: A reinvestigation of the function of the mammalian urinary bladder. Am J Physiol 232 (Renal Fluid Electrolyte Physiol 1):F187-F195, 1977 43. Taylor A, Windhager EE: Possible role of cytosolic calcium and Na-Ca exchange in regulation of transepithelial sodium transport, Am J Physiol 236 (Renal Fluid Electrolyte Physiol 5):F505-F512, 1979 44. Linshaw M: Effect of metabolic inhibitors on renal tubule cell volume. Am J Physiol 239 (Renal Fluid Electrolyte Physiol 8):F571-F577, 1980 45. Reuss L: Electrical properties of the cellular transepithelial pathway in Necturus gallbladder: III. Ionic permeability of the basolateral cell membrane. J Membrane Biol 47:239-259, 1979 46. Shindo T, Spring IGR: Chloride movement across the basolateral membrane of proximal tubule cells. J Membrane Biol 58:35-42, 1981