AJP CENTENNIAL
A century of (epithelial) transport physiology: from vitalism to molecular cloning

Stanley G. Schultz

Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77225

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During the past century, the generally accepted view of the function of biological membranes has evolved from that of a static, inert lipoprotein envelope that simply separates the intracellular machinery from the outside milieu, to that of a dynamic structure that is, in fact, an integral part of that machinery actively involved in homeostasis and bioenergetics. This brief review traces that paradigm shift with special emphasis on the evolution of central concepts in epithelial transport physiology.

history; membrane physiology; absorption; secretion

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... there is present in the living skin of the frog a vital absorptive force dependent upon protoplasmic activity and comparable to the vital secretory force of glands, and that by virtue of this vital action the skin is actually able to cause a stream of fluid to pass from its outer surface to its inner surface ... . These phenomena failed to manifest themselves when dead skin was used for experiment instead of that which had been but recently removed and which was therefore still in a living condition (44).

But for a few words and phrases here and there, I would not have been surprised to see the above statement in a paper published in the American Journal of Physiology about midcentury (1955-1965) when the story of fluid transport by epithelial tissues, in vitro, was beginning to unfold. However, that statement was published more than a century ago, in 1892 to be exact, by E. Waymouth Reid in the British Medical Journal under the title "Report on experiments upon absorption without osmosis" (44). Reid, who was a Lecturer of Physiology at University College Dundee, used the apparatus shown in Fig. 1 to demonstrate that fluid is transferred from the solution bathing the outer surface of isolated frog skin to the solution bathing its inner surface in the absence of an external osmotic driving force, provided the skin is viable. He even short-circuited the frog skin potential to rule out the possibility that fluid flow was driven by electroosmosis, a mechanism suggested in 1872 by Engelmann (15).


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Fig. 1.   Apparatus employed by Reid for the study of transepithelial fluid movement (absorption and secretion) in the absence of an external osmotic pressure difference. Each hemichamber was equipped with a thermometer (C) and capillary tube (D) for the study of volume displacements. [From Reid (44).]

In a later series of studies, published in 1901 (45), Reid demonstrated that ileum removed from a rabbit "in full digestive activity," and mounted as a flat sheet in the apparatus shown in Fig. 1, was capable of transferring fluid from the luminal bathing solution to that bathing the serosal surface of the tissue; but, on "... a piece of ileum from a rabbit which as a result of a chill was suffering from diarrhoea ... the motion of fluid is in the reverse direction." He also showed that pilocarpine could reverse the fluid movement across isolated rabbit ileum from the absorptive to the secretory direction. Absorption as well as secretion could take place in vitro in the absence of external osmotic pressure differences!

Reid had clearly and, to the best of my knowledge, for the first time unambiguously demonstrated and recognized "active transport" by an in vitro preparation; that is, the flow of matter in the absence of an external (conjugate) driving force that was dependent upon a source of metabolic energy!

However, what should have been a clarion call heralding a major conceptual breakthrough in epithelial biology turned out to be barely a whimper. The significance of Reid's observations went largely unnoticed. He is not mentioned at all in the 1943 or 1952 editions of Davson and Danielli's classic book The Permeability of Natural Membranes (9), which has an entire chapter dealing with epithelial absorption and secretion, and his observations are largely dismissed by Höber (23) in the 1945 edition of Physical Chemistry of Cells and Tissues.

Why? Could it be because he used the phrase "vital force" to describe his observations---a phrase that was perhaps the naughtiest in the naturalist's lexicon during that era?

Indeed, it is likely that it was the taint of vitalism that cost Galvani dearly in his celebrated debate with Volta 100 years earlier. As discussed by Pera (43) in his delightful and penetrating monograph entitled The Ambiguous Frog, "Galvani was intent on explaining (muscle) contractions iuxta principia biologica; that is, on confirming the biological origin of the frog's current: Volta, instead, on explaining these and other phenomena iuxta principia physica; that is, on establishing the physical origin of all currents." Neither of the contestants disputed the experimental findings (hence the word "ambiguous" in the title)! How could the notion of "animal-generated electricity"---smacking as it does of an elan vital---compete with Volta's widely accepted demonstration that electrical current can be generated by the contact of dissimilar metals (the Voltaic pile)? Despite the fact that Galvani was correct, his position was rejected by the scientific community shortly after his death, while Volta went on to fame and fortune (43).

As discussed by Mayr (34, 35), from the time of the Scientific Revolution, pioneered by Descartes, Galileo, Kepler, and Newton, until relatively recently, there had been an ongoing, often heated, debate between the "physicalists" and the "vitalists" over the explanation of phenomena that are unique to living organisms, the former confident that these phenomena would succumb to physicochemical explanations and the latter denying that faith. And it is difficult to imagine a worse time to even hint at the possible involvement of vital forces in transport phenomena than the closing decades of the 19th century and the opening decade of the 20th. It was during that period that Nernst (1888) and Planck (1900) extended the earlier considerations of Fick (1855) and derived the equation for electrodiffusion that dominates our thinking to this day; van't Hoff (1887) developed the law for osmotic flow across semipermeable membranes; and Gibbs (1876) and Donnan (1911) independently described the equilibrium condition that bears their names. Physics, dominated by Newtonian mechanics, Maxwellian electrodynamics, and thermodynamics, was reigning supreme, and the physicalist's approach to the understanding of the functions of living organisms was dominant. While it is not clear whether E. Waymouth Reid was an "orthodox vitalist" or whether he used the phrase "vital absorptive forces" loosely, it seems that his thesis never had a chance!

My goal in this historical review is to start where Reid left off and provide a bird's-eye view of the evolution of seminal concepts in epithelial transport during the past century. While the focus will be on epithelia, it is clearly impossible to divorce that subject from transport and membrane physiology in general, hence the parentheses in the title. Furthermore, my choice of the word "evolution" instead of "history" is quite deliberate; a history of the subject would necessarily consume volumes. Therefore, my focus is on the emergence of concepts that have shaped our thinking, our research, and our interpretations of the results of research and that have delivered us to the current state of the art. In the sense of Thomas Kuhn (29), I try to trace the evolution of the paradigms that have defined epitheliology and its scientific community. I do this with some degree of trepidation, because not all of us will necessarily agree with my emphases and biases; I apologize in advance for any slights---none are intended.

The Period of Passivity (1895-1940)

The period commencing 1885 and lasting for more than four decades was dominated by the view that the plasma membrane is, for the most part, an inert envelope; this was the "paradigm" that was embraced by the community of membrane physiologists and, in turn, defined that community. It was the "normal science," firmly founded on the triumphs of the contemporary physical chemistry and thermodynamics, that shaped ongoing research and the interpretation of experimental results.

This period opens with the classic work of Overton (see Ref. 27), showing that the ease with which a large number of solutes permeate cell membranes is closely correlated with their olive oil-to-water partition coefficients (i.e., lipid solubility). Twenty-five years later, Gorter and Grendel (20) demonstrated that there is sufficient lipid in the erythrocyte membrane to form an envelope consisting of a bimolecular layer, and shortly thereafter Danielli and Davson (8) proposed the lipoprotein model that gripped the field at least until the middle of this century. This model, illustrated in Fig. 2, was the Gorter-Grendel bimolecular lipid construct embellished by adsorbed protein layers at each lipid-water interface. Could anything appear more inert?


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Fig. 2.   Davson-Danielli model of the cell membrane. [From Danielli and Davson (8).]

The notion that solutes equilibrate across biological membranes by simple diffusion was so pervasive, that, when Collander and his associates (cf. Refs. 5, 8, 23) noted that some small hydrophilic solutes crossed membranes more rapidly than could be predicted on the basis of their lipid solubilities alone, they suggested that the membrane behaves both as a lipid barrier and a molecular sieve; apparently no serious consideration was given to mechanisms of permeation other than simple diffusion. The only exception to this generalization was for the case of ions, in which Osterhout (40) and Lundegårdh (32) suggested that permeation may be mediated by reversible formation of a complex with an oppositely charged membrane component that serves as a transmembrane ferry. Suffice it to say that the word "carrier" cannot be found in the index of the Davson and Danielli book (9).

To be sure, many instances were noted "... where the laws of thermodynamics are apparently broken and molecules accumulate on one side of a membrane, in excess of the amount on the other side" (Ref. 9, p. 1). Furthermore, since the improbability of violating the Second Law of Thermodynamics was generally appreciated, it was clear that these "anomalous" transport processes involved an investiture of metabolic energy on the part of the cell. However, the notion of making the membrane the site where energy was coupled to the vectorial flow of matter---to endow the membrane with a "Maxwell demon"---seemed anathema (9, 23).

For example, it was well established that certain sugars were capable of moving across small intestinal mucosa more rapidly than others and that renal tubular epithelial cells were capable of completely extracting glucose from the glomerular filtrate, thereby rendering the final urine sugar free, and that these processes could be blocked by inhibitors of phosphorylation. A widely accepted explanation for these observations (65) was that the apical and basolateral membranes of these epithelial cells are freely permeable to sugars (glucose and galactose) but not to their phosphorylated esters. Thus the free sugars enter the cell by simple diffusion across the apical membrane, whereupon prompt phosphorylation prevents back-diffusion. This maintains the concentration gradient for entry and permits accumulation of the ester within the cell to potentially high levels. Dephosphorylation of the ester at or near the basolateral membrane then permits the free sugar to exit the cell. Without dissecting the many shortcomings of this model, it should be clear that the cell membranes were portrayed as exclusively passive barriers.

An even starker example of the "period of passivity" was the explanation for the asymmetric distribution of Na+ and K+ across the plasma membrane of muscle championed by E. J. Conway, one of the most influential figures in the field at that time, and P. J. Boyle (2). These investigators proposed that the cells were "born" with a low intracellular Na+ concentration and remained that way because their membranes were absolutely impermeable to that cation (which has a larger hydrated radius than K+ and Cl-). In addition, the membranes were permeable to K+ and Cl-, which then become distributed in accordance with a "double Donnan equilibrium," taking into account the high concentrations of organic anions that are also trapped within the cell. This model was completely consistent with the prevailing view of the "inert membrane"; it invoked no vital forces whatsoever, and it was hailed by J. F. Danielli as "very promising" (Ref. 9, p. 269).

In conclusion, as any reader of the first (1943) edition of The Permeability of Natural Membranes (9) will quickly perceive, the first four decades of this century were dominated by a minimalist approach to membrane transport physiology that is captured by the static membrane model illustrated in Fig. 2. Although "nonpassive" transport phenomena were certainly recognized, they were treated as mysteries awaiting further study with new techniques and, hopefully, explanations that do not invoke vital forces. It seems as if every possible effort was made to preserve the prevailing inert membrane paradigm.

Isotopes and the Birth of a New Paradigm (1940-1960)

While the period 1895-1940 was dominated by a view of the plasma membrane as an inert barrier and a focus on "passive permeability," that paradigm was discarded and replaced during the two decades that followed, thanks, in large part, to the availability of radioactive isotopes and their application to the study of membrane transport.

No sooner had Conway published his double Donnan equilibrium model centered on the notion that the low intracellular Na+ concentration in muscle was an "inherited property" sustained by a membrane that was impermeable to that cation, than the work of Heppel, Fenn, Steinbach, Harris, and others, recounted in a recent review by Robinson (47), showed that, under certain conditions, cells may gain Na+ and lose K+ reversibly, and seriously challenged Conway's model. And, in an insightful analysis of the available data, published in 1941, Dean (10) concluded that the asymmetric distribution of Na+ and K+ across the muscle membrane required a mechanism that extrudes Na+ from the cell, i.e., a "pump."

As pointed out by Robinson (47), the invocation of a Maxwell demon---a mechanism that could direct traffic across a membrane in a direction contrary to that predicted by the Second Law of Thermodynamics---was not enthusiastically embraced by the community of physiologists. Such a mechanism had no biochemical precedent or anlage. How could such a demon be reconciled with the "accepted" model of membrane structure (Fig. 2)? Some suggested alternative explanations such as the "association-dissociation" model proposed by Ling (31), which rejected the role of the cell membrane and argued that Na+ and K+ are adsorbed to sites, in the cytoplasmic gel, whose selectivity for these cations is influenced by the metabolic state of the cell; regrettably, some on the fringe adhered to this view long after it was discredited by the findings discussed below.

Indisputable evidence for a Na+ pump and active transport came from the brilliant studies of Hans Ussing on isolated frog skin. In the mid-1930s, August Krogh, Ussing's mentor, together with Hevesy and Neils Bohr, convinced the Rockefeller Foundation that radioactive isotopes would be a boon to biomedical research, and the Foundation contributed to the construction of the first cyclotron in Europe at the University of Copenhagen, the Bohr Institute (41, 49). The accelerator was completed in 1938 and, in the early 1940s, while Denmark was still occupied by German troops, Krogh, Hevesy, and their co-workers set the stage for the modern era of membrane physiology. With the use of radioactive isotopes, they demonstrated that cations and anions readily exchange across animal and plant cell membranes. In his Croonian Lecture of 1946, Krogh stated: "The main object of this paper is to discuss the large differences in concentrations of individual ions between the interior of living cells and the fluid surrounding them, to bring out the point that such differences are normally and perhaps always brought about and maintained by some special activity on the part of the cell, while diffusion processes are all the time tending to reduce them" (cf. Ref. 49). Krogh clearly anticipated the currently accepted view of the interactions between pumps and dissipative processes (leaks) in maintaining the steady state. His phrase "special activity" was Reid's "vital force," which was soon to evolve into "active transport"!

In 1945 Krogh retired, and Hans Ussing picked up where his mentor left off. In 1948, he and Levi (30) demonstrated 24Na+ exchange across the plasma membrane of frog sartorius muscle and coined the term "exchange diffusion." A year later he published "The distinction by means of tracers between active transport and diffusion" (64), in which he derived the "flux-ratio" equation1 and defined the criteria for passive transport. In 1951 came his crowning triumph with Zerahn (65), the demonstration of the equivalence between the short-circuit current and active Na+ transport across isolated frog skin. Na+ could be transported across viable frog skin from the outer bathing solution to an identical inner bathing solution in the absence of external driving forces. The use of tracer permitted an entirely "noninvasive" approach, and the results could not be attributed to different properties of the two bathing solutions.

In contrast with Reid's experience 50 years earlier, Ussing's demonstration of active transport was immediately embraced, and a new paradigm was born.

Soon thereafter, Gárdos (19), with the use of a crude preparation of resealed red blood cell "ghosts," demonstrated the "active" uptake of K+, and, independently, Hoffman (24), using a much cleaner preparation reasonably free of cytoplasmic contaminants, demonstrated the "active" extrusion of Na+, provided that the ghosts contained ATP or appropriate metabolic precursors; similar findings were reported, in 1957, for squid axon by Caldwell and Keynes (3). About the same time, Zerahn (71) demonstrated a linear relation between the rate of Na+ absorption by frog skin and the rate of O2 consumption. Thus the Second Law of Thermodynamics is preserved! There is no reason to invoke a Maxwell demon! Reid's vital absorptive force is ATP!

But, by what agency? Enter Skou, a modest Danish surgeon who, in 1957, published a paper with a most unpretentious title, "The influence of some cations on an adenosinetriphosphatase from peripheral nerves" (61). The "... some cations ..." were Na+, K+, and Mg2+! Skou had identified an ATPase activity in the membrane of crab nerve whose activity, in the presence of Mg2+, was maximum in the combined presence of Na+ and K+ and speculated that this might be Dean's pump---a notion made much more explicit in a later publication (62). Skou had discovered an enzyme that has proved to be one of the most ubiquitous, and intensively studied, membrane-bound proteins in higher animals, an enzyme that is now synonymous with the Na+-K+ pump.2

Thus, by the end of the 1950s, the results of numerous studies on nonepithelial cells, such as erythrocytes, muscle, and nerve, many made possible by the availability of radioactive isotopes, led to the near-universally accepted notion that the high intracellular K+ and low intracellular Na+, characteristic of virtually all cells in higher animals, are the result of a steady state created by interactions among the Na+-K+ pump and dissipative pathways or "leaks" for Na+ and K+; the nature of these leak pathways was still a matter of conjecture, although a number of investigators had already raised the specter that there might be "pores" or holes through membranes (cf. Ref. 22)!

The stage was thus set for the birth of the modern era of epithelial transport physiology with the introduction of the Koefoed-Johnsen-Ussing (KJU) double-membrane model (28). These investigators demonstrated that, in the presence of an impermeant anion, the outer membrane of isolated frog skin behaves like a "Na+ electrode" (and was therefore permselective for that cation), whereas the inner membrane behaves like a "K+ electrode" (and was therefore permselective to that cation). The now classic double or series membrane model (Fig. 3) could, at one and the same time, account for the high intracellular K+ and low intracellular Na+ activities characteristic of cells from all higher animals, active transcellular Na+ transport, the equivalence between active Na+ transport and the short-circuit current, and the origin of the frog skin potential---a problem that had perplexed physiologists since the time of DuBois-Reymond (14). All one had to do was rearrange transport processes known to exist in nonepithelial cells---no new processes unique to epithelial cells had to be invoked (or so it seemed at the time). The model fulfilled all the desiderata of esthetics and parsimony that attract scientists and was instantly embraced by the scientific community. Another paradigm was born, in this case de novo, since there was none to be replaced.


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Fig. 3.   Koefoed-Johnsen-Ussing double (series) membrane model for Na+ absorption by isolated frog skin. Ocm and Icm denote the outer and inner cell membranes, respectively. [From Koefoed-Johnsen and Ussing (28).]

The "Fleshing-Out" of the Double-Membrane Model (1960-1980)

In the two decades that followed the introduction of the KJU model, epithelial transport physiology rapidly grew from infancy to adulthood. The double-membrane concept and the basolateral localization of the Na+-K+ pump was extended to a wide variety of absorptive and secretory epithelial cells and was not found wanting. If any single thread ran through this maturation period it was "gradient coupling"; in other words, the finding that membrane-bound carriers or "porters" can couple the flows of two or more solutes with the final direction of flow(s) determined by the "cumulative thermodynamic gradient" or the net electrochemical force. This remarkable notion was clearly embedded in what was, at the time it appeared, one of the most unorthodox, if not apostate, speculations in the past 50 years, namely, Peter Mitchell's "chemiosmotic" hypothesis. In his now classic paper, published in Nature in 1961, Mitchell (36) wrote "The underlying thesis of the hypothesis put forward here is that if the processes that we call metabolism and transport represent events in a sequence, not only can metabolism be a cause of transport, but also transport can be the cause of metabolism. Thus we might be inclined to recognize that transport and metabolism, as usually understood by biochemists, may be conceived advantageously as different aspects of one and the same process of vectorial metabolism." In a later paper (37), Mitchell, who was not averse to neologisms, introduced the terms "uniport," "antiport," and "symport" (for co- and countertransport) and "primary" and "secondary" translocation, to classify types of gradient coupling. The bottom line, which found formal expression in the language of irreversible thermodynamics (51), is that carriers recognize bulk electrochemical potential differences. But, as recently pointed out by Kaback (26), "... the molecular mechanism(s) by which free energy stored in such gradients is transduced into work or into chemical energy remains enigmatic."

The notion of gradient coupling first emerged explicitly from the elegant studies of Robert Crane and his co-workers (6) on the mechanism of sugar absorption by small intestine. Crane was clearly the first to propose, explicitly, that the uphill movement of an organic solute (in this case sugars) could be energized by coupling to the downhill movement of Na+, but he failed to appreciate the connection between the "Na+ gradient hypothesis," the KJU model, and transepithelial Na+ transport. A few years later, Schultz and Zalusky (58, 59), applying the short-circuit technique to the study of mammalian small intestine (rabbit ileum), demonstrated the cotransport of Na+ with sugars and amino acids and, as illustrated in Fig. 4, proposed a synthesis of Crane's hypothesis and the KJU "double-membrane model" (58). Within the next decade, additional Na+-coupled apical entry mechanisms were uncovered, resulting in the evolution of double-membrane models of Na+-absorbing epithelial cells to the current state illustrated in Fig. 5. Thus there appear to be four general mechanisms that mediate Na+ entry into these absorptive cells: a) diffusion through channels (uniporters) that can be blocked by the diuretic, amiloride; b) coupling to the entry of a large number of solutes, many having distinct carriers (e.g., sugars, several classes of amino acids, bile salts in the ileum, phosphate, some vitamins, etc.); c) coupling to the entry of Cl-, most often, but not always, in the form of a "tritransporter" involving one Na+, one K+, and two Cl-; and d) coupling to the countertransport of H+. The properties of these mechanisms have been reviewed exhaustively and are not the purview of this historical review. The important message is that the synthesis of the Na+ gradient coupling hypothesis and the double-membrane model, with the basolateral localization of the primary active transporter (the Na+-K+ pump) responsible for establishing and maintaining the transapical gradient, led to a unified framework that could describe transport across a wide variety of Na+-absorbing epithelia; the double-membrane model with its basolateral pump became a framework that could be embellished with any number of co- or countertransporters from a large menu (53) to accommodate experimental observations. The results of studies at the subcellular (e.g., membrane vesicles) and molecular levels have, without exception, confirmed the asymmetric localization of transporters central to this model and, while there still remain some loose ends to be tied up, this paradigm for transcellular transport appears to be established beyond doubt.


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Fig. 4.   Schultz-Zalusky model for coupling between transepithelial Na+ and sugar (S) transport by small intestine. The phlorizin-sensitive apical cotransporter is now known to be SGLT (70). The mechanism responsible for sugar exit from the cell across the basolateral membrane was not known at that time; it is now known to be facilitated diffusion mediated by the protein GLUT-2 (25). [From Schultz and Zalusky (57).]


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Fig. 5.   Composite model of Na+-absorbing epithelial cells. Note that this figure only depicts the different mechanisms (a-d; see text) that can mediate Na+ entry into the cell across the apical membrane of various epithelial cells. Other transporters found in the apical and basolateral membranes of epithelial cells are discussed elsewhere (53). S, solutes.

The period 1960-1980 also witnessed the development of the generally accepted model for NaCl secretion by some epithelial cells shown in Fig. 6. This model emerged from the convergence of studies on such seemingly disparate subjects as human cholera, the rectal gland of the shark, the "Cl- cell" of fish gills, cornea, and the nasal gland of marine birds and has most recently received major impetus by attempts to understand the secretory defects associated with cystic fibrosis (17, 67). Once again, the general framework is the KJU double-membrane model featuring a Na+ gradient-driven tritransporter, in this instance at the basolateral membrane, and a regulated Cl- channel at the apical membrane; the best understood of these Cl- channels is the cystic fibrosis transmembrane conductance regulator (CFTR), whose activity is regulated by adenosine 3',5'-cyclic monophosphate-dependent protein kinase A activity. A less-well-understood, Ca2+-activated Cl- channel is likely to be the one responsible for Reid's observation that pilocarpine stimulates fluid secretion by rabbit ileum (67).


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Fig. 6.   Model for Cl- secretory cells. Basolateral membrane features the K+-Na+-2Cl- tritransporter, the Na+-K+ pump, and at least 1 type of K+ channel for recycling of that cation. Also shown is a basolateral membrane receptor for a secretagogue (S) that is usually coupled by G protein to adenylate cyclase. It should be noted that apical membrane Cl- channels other than and/or in addition to cystic fibrosis transmembrane conductance regulator (CFTR) may mediate Cl- exit from the cell across that barrier. Na+ follows transcellular Cl- secretion via the paracellular route. PKA, protein kinase A.

During the same period of time, the role of Na+ gradient coupling became more and more widely recognized as a major mechanism of secondary active transport in all eukaryotic cells, and, beginning with the observations of West in 1970 (68), H+ gradient coupling emerged as the major mechanism for secondary active transport in prokaryocytes (26, 38).

Thus, within a relatively brief period, two general phenomena that form the foundation of our current understanding of membrane transport were established beyond reasonable doubt, namely, the ability of cell membranes to establish ion gradients ("proton-motive" and "natrio-motive" forces) at the expense of ATP ("primary active transport") and their ability to utilize the potential energy stored in those ion gradients to energize the uphill movement of a co- or countertransported solute ("secondary active transport").

Another feature of epithelial transport that surfaced during this remarkable 20-year period was that transepithelial solute or solvent flow is not restricted to the transcellular pathway, requiring movements across the two limiting membranes arranged in series, but can also traverse low-resistance "paracellular" pathways that circumvent the cells. This notion arose from observations that, unlike the frog skin and toad urinary bladder, a number of Na+-absorbing epithelia are characterized by low transepithelial electrical potential differences and resistances. While the former could (and can) be attributed to electroneutral Na+-coupled entry mechanisms, it was immediately apparent that the latter was almost certainly due to the fact that ions can somehow flow between cells (57, 69). The unequivocal identification of these pathways with the junctional complexes that bind the epithelial cells together at their apical ends---which were unfortunately named zonulae occludens (i.e., occluding or tight junctions) by their discoverers (16)---by Frömter and Diamond (18), opened a new area, an area that has become increasingly vibrant in recent years with the recognition that the permeability characteristics of these junctional complexes are not static but may be regulated under physiological and pathophysiological conditions (cf. Ref. 33).

Finally, the 1960-1980 decades also witnessed the emergence of the now widely accepted explanation for fluid absorption by epithelia. By 1965, there were three sets of observations that any model of fluid absorption had to accommodate, namely, 1) water absorption may take place in the absence of or against a transepithelial osmotic, or hydrostatic, pressure difference; 2) water absorption, under those conditions, is dependent on the presence of active solute transport; and 3) under many conditions, the absorbed fluid is isotonic, within experimental limits, with the fluid bathing the mucosal surface of the tissue (53). A conceptual model, founded on the principles of irreversible thermodynamics, that could account for these observations was proposed by Curran and McIntosh (7) and is illustrated in Fig. 7A. Briefly, solute is transported across membrane alpha  from compartment 1 into compartment 2, rendering the latter hypertonic. If the reflection coefficient of membrane alpha  for the solute (i.e., sigma alpha ) is greater than that of membrane beta  (i.e., sigma beta ) then, as discussed elsewhere (50, 54), fluid can be transferred from compartment 1 to compartment 3 in the absence of, or against, an osmotic pressure difference. This model, in my opinion, represents the high point of the application of irreversible (or nonequilibrium) thermodynamics to membrane transport. The phenomenon of "absorption without osmosis," first proclaimed by Reid and later confirmed by many others, could finally be explained in principle, and the problem devolved into identifying the biological analogs or counterparts of the Curran-McIntosh double-membrane model. As I discussed previously (50), the "standing osmotic gradient" model proposed by Diamond and Bossert (12) did just that (Fig. 7B) and, while recent developments have relieved the need for some of the restrictions of the original model, the basic premise, namely, that water flow is energized by local osmotic differences, appears solid (63). Finally, before leaving the subject of water absorption, it is of interest to reflect on an earlier model proposed by Hartley in 1937 (21) to explain water absorption by the renal tubule, leading to the elaboration of a concentrated urine. According to this model (Fig. 7C), compartment 2 is rendered hypertonic to compartment 1 by the continual production of a diffusible metabolite; this draws water from compartment 1 across membrane B, thereby concentrating the salt contained in that compartment. If, instead, Hartley had invoked the active transport of the solute from compartment 1 to compartment 2, mediated by a Maxwell demon in membrane B, he would have anticipated the Curran-McIntosh model; but that, of course, was not the "normal science" in 1937, and his notion might have suffered the fate of E. Waymouth Reid's!


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Fig. 7.   A: Curran-McIntosh double-membrane model for solute-coupled water transport (7). B: model of an epithelial cell indicating possible anatomic candidates for the compartments and barriers in the Curran-McIntosh model. BB, brush border; ZO, tight junction; C, underlying capillary. According to the Diamond and Bossert model (12), solute is deposited into the intercellular space (2) close to the ZO, and water drawn into compartment 2 establishes a "standing osmotic gradient." More recent studies (63) suggest that this feature of the original model is unnecessary; solute may be deposited anywhere in the intercellular and subepithelial spaces and come to close osmotic equilibration with water drawn from the cell across the basolateral membranes. [From Schultz and Curran (54).] C: model proposed by Hartley (21) for concentration of a salt solution where the energy for this process is derived from the metabolic production of an osmotically active solute in compartment 2. [From Davson and Danielli (9).]

Before concluding this section, let us turn our attention to parallel developments that took place in the 1960s, in an entirely different discipline, which revolutionized our view of membrane structure. While physiologists were embellishing cell membranes with purported carriers and "channels" (or pores), all of which were largely inferential, biochemists and biophysicists were uncovering the roles of hydrophobic bonding in protein-protein and protein-lipid interactions. As pointed out by S. J. Singer (60), it soon became clear that the Davson-Danielli-Robertson (DDR) model (46), which viewed the membrane as a "... railroad-track assembly with a phospholipid bilayer sandwiched between two layers of unfolded protein sheets ..." (60) was thermodynamically untenable, as was any flip-flopping of proteins across the membrane as pictured by "ferry boat" models of membrane carriers. To accommodate these new findings and remedy the shortcomings of the "classical" (DDR) membrane model, Singer and his co-workers proposed the "fluid mosaic model" (Fig. 8), which featured proteins embedded in the lipid bilayer, some of which span the thickness of the membrane ("integral proteins") and thus afford avenues of direct communication between the surrounding aqueous compartments. This new view lent biochemical and structural credence to the "cartoons" being imagined by physiologists, cartoons that were soon to acquire molecular identities.


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Fig. 8.   A: Davson-Danielli-Robertson lipoprotein membrane model. B: fluid-mosaic membrane model. [From Singer (60).]

The Dawning of a New Era

By the early 1980s the double-membrane model had successfully weathered all challenges, and it, together with its apical and basolateral membrane embellishments, successfully accommodated an enormous body of phenomenology at the descriptive level. The stage was thus set for the two general avenues of investigation being pursued by investigators in epithelial physiology today: one reductive and directed at elucidating the workings of transporters at the molecular level and the other integrative and aimed at gaining insight into the biology of the transporting epithelial cells and tissues.

History will undoubtedly record the final two decades of the 20th century as the period during which the combination of investigator creativity, the development of and advances in molecular biological and patch-clamp technologies, and the gracious hospitality of Xenopus laevis oocytes resulted in an explosive leap forward in our identification and understanding of "transporters" at the molecular level. Most of the carriers and channels deduced from functional studies, which annotate cartoons of epithelial cell apical and basolateral membranes, have gained molecular identities and thus scientific credibility. They are, as was suggested by Singer (cf. Ref. 60), integral proteins with alpha -helical membrane-spanning segments that hydrophobically anchor the protein to the membrane. The apical membrane amiloride-sensitive Na+ channel (4), Na+-coupled sugar carriers (70), K+-Na+-2Cl- tritransporter (39), Na+-Cl- cotransporter (39), and Na+/H+ countertransporters (13) of Na+-absorbing epithelial cells (Figs. 3 and 4) have been cloned, sequenced, mutagenized, expressed in heterologous systems, and studied extensively as have various isoforms of the Na+-K+-ATPase at their basolateral membranes. Water-selective channels, appropriately termed "aquaporins," have been identified in the apical and/or basolateral membranes of a number of these epithelial cells (48), thus providing pathways through which water and solute flows can be linked by local osmotic pressure differences. CFTR, the culprit in nature's "knockout experiment," cystic fibrosis, has likewise been scrutinized at the molecular level, yielding unparalleled insight into one of the most prevalent of human congenital afflictions (67), and on and on.

Space limitations do not permit me to catalog, comprehensively, all of the transporters that have been identified at the molecular level; suffice it to say that there are many, and it is not unrealistic to predict that in the very near future the catalog will be complete. It is remarkable that virtually every transporter that was conjectured from the results of functional studies using traditional approaches, often seemingly figments of active imaginations, has acquired a molecular identity; in this respect there is a remarkable complementarity between the "old biology" and the "new biology." Undeniably, this complementarity is in part tautological, because many transporters have been cloned using techniques that select for the expression of specific functional characteristics; however, many have not, and the correspondence remains impressive.

There is a growing body of information dealing with the behavior and regulation of these transporters expressed in heterologous systems. Although much of this information appears to parallel the behavior of these transporters in their native environments, care still must be exercised in extrapolating from foreign systems to the parent cell; Xenopus oocytes are not epithelial cells. Evidence for "promiscuous coupling" where unexpected properties emerge following the simultaneous expression of two different transporters is surfacing, forewarning that the "whole" may be more than the simple sum of its parts. In addition, while the results of ingenious studies employing site-directed mutagenesis have provided seductive glimpses into possible structure-function relations, it seems unlikely that a definitive understanding of the workings of these transporters at the molecular level will be achieved without knowledge of tertiary structure. Until recently, however, most integral membrane proteins have defied high-resolution crystallographic approaches. The recent success in obtaining X-ray structure of bacteriorhodopsin at 2.5 Å resolution from three-dimensional crystals grown in a lipid cubic phase offers great promise for the future (42). Indeed, it is not too much of a fantasy to envision a time in the not-too-distant future when the physicalist's dream of understanding transport processes in mechanical terms at the molecular level will be realized.

In parallel with studies at the molecular level, there are ongoing efforts to arrive at a comprehensive understanding of how epithelial cells do their jobs and survive. These cells, be they absorptive or secretory, are, with few exceptions, the interfaces between the interstitial fluid compartment and the outside world. As such, they are the guardians of Claude Bernard's "milieu intérieur" whose stability or "fixité" is an "... essential condition for a free and independent life" (1). As pointed out earlier (52, 56), there are no cells in the body whose intracellular composition and volume are more assaulted and threatened during the course of their normal activities. How these cells respond to extracellular and intracellular signals, how the activities of transporters are coordinated by feedback and cross talk mechanisms, how transporters are shuttled to and from apical and basolateral membranes "on demand," and how the genetic apparatus is prodded to upregulate or downregulate transporters in response to changing needs are but a sampling of questions that have surfaced in recent years and that await closure at the cellular and molecular levels.

Finally, much of the focus of the science of (epithelial) transport physiology during the past century has been on "what" and "how" questions; in other words, on questions of proximate causation. As recently discussed by Diamond (11) and Mayr (35), there are also the "why" questions, or questions of ultimate causation, that deal with the evolutionary biology and design of organ systems. Thus, for example, while cell and molecular biology will ultimately provide us with a proximate explanation for the maximum capacity of the human kidneys to reabsorb filtered glucose per unit time in terms of the density of SGLT carriers in the apical membranes of renal proximal tubule cells, their affinity for binding glucose, the turnover number of these carriers, the Na+ gradient across the apical membrane, and so forth, these are answers to "what" and "how" questions. The ultimate explanation for why the tubular maximum of human kidneys is what it is---a perfectly legitimate concern of epithelial transport physiologists---will require, in addition, considerations of human physiology and pathophysiology, natural selection, and principles of biological design. To quote from Jared Diamond (11): "The explosion of cell and molecular biology in recent decades has produced a large body of descriptions and of proximate mechanistic explanations, but few ultimate explanations and little integration into a larger intellectual context. The missing broader context must come from combining understanding of evolutionary principles to arrive at a quantitative explanation of biological design. Such explanations will constitute a science of evolutionary physiology."

Viewed in this light, the past century of (epithelial) transport physiology is but a prelude of things to come.

    ACKNOWLEDGEMENTS

I am grateful to Drs. Joseph F. Hoffman and Ernst Knobil for their careful reading of this manuscript and their helpful suggestions.

    FOOTNOTES

1 As discussed elsewhere (51), the flux-ratio equation had been derived earlier by Behn (1897) and, contemporaneously, by Teorell (1949). However, Ussing must be credited with recognizing the power of this equation for the description of uncomplicated electrodiffusion of isotopic tracers across biological membranes and the practical application of this theoretical approach.

2 In the interest of brevity, I have focused on the "Na+ side" of the pump. Robinson (47) has briefly reviewed the evidence leading up to the notion of obligatory coupling between the flows of Na+ and K+ in opposite directions.

Address for reprint requests: S. G. Schultz, Dept. of Integrative Biology and Pharmacology, Univ. of Texas Medical School, PO Box 20708, Houston, TX 77225.

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