Following Ussing’s legacy: from amphibian models to mammalian kidney and brain

Abstract

Professor Hans H. Ussing (1911–2000) was one of the founding members of the field of epithelial cell biology. He is most famous for the electrophysiological technique that he developed to measure electrogenic ion flux across epithelial tissues. Ussing-style electrophysiology has been applied to multiple tissues and has informed fields as diverse as amphibian biology and medicine. In the latter, this technique has contributed to a basic understanding of maladies such as hypertension, polycystic kidney disease, cystic fibrosis, and diarrheal diseases to mention but a few. In addition to this valuable contribution to biological methods, Prof. Ussing also provided strong evidence for the concept of active transport several years before the elucidation of Na⁺K⁺ATPase. In addition, he provided cell biologists with the important concept of polarized epithelia with specific and different transporters found in the apical and basolateral membranes, thus providing these cells with the ability to conduct directional, active and passive transepithelial transport. My studies have used Ussing chamber electrophysiology to study the toad urinary bladder, an amphibian cell line, renal cell lines, and, most recently, choroid plexus cell lines. This technique has formed the basis of our in vitro mechanistic studies that are used in an iterative manner with animal models to better understand disease progress and treatment. I was honored to be invited to deliver the 2022 Hans Ussing Lecture sponsored by the Epithelial Transport Group of the American Physiological Society. This manuscript is a version of the material presented in that lecture.

BACKGROUND: PROFESSOR USSING’S LEGACY

It is a humbling experience to be granted the honor of giving a Ussing Lecture since Hans H. Ussing’s ground-breaking work has formed the basis of my research career as an epithelial cell physiologist. Those of us who use Ussing-style electrophysiology in our research are fond of telling our students that this technique was first developed to study the absorption of Na⁺ across frog skin, thereby allowing the amphibian to extract this important nutrient from pond water. However, in delving deeper into the history of Dr. Ussing’s career, one is struck by his truly outstanding contributions to concepts associated with transcellular ion flux that we now take for granted. A more comprehensive view of Prof. Ussing’s life and contributions can be found in an excellent paper by Prof. Erik Larsen, one of his colleagues at the University of Copenhagen (1).

To fully appreciate Ussing’s contributions, one must go back in time and imagine when epithelial cells were black boxes; polarization of transporters to either the apical or basolateral membrane was unknown, the concept of active transport was controversial, energy-requiring transporters such as Na⁺K⁺ATPase were yet to be elucidated, and radiolabeled tracers were a new and exciting tool in a transport physiologist’s armamentarium. Ussing’s mentor and colleague, August Krogh, had suggested the concept of active transport and put forward the question of whether organisms invest metabolic energy in creating and maintaining ion concentration gradients. The advent of radiolabeled tracers and, in part, work in collaboration with Niels Bohr, strengthened some of the hypotheses that had been proposed. Ussing was in the unique position to combine a variety of techniques to address the important issue of active transport. The frog skin was chosen as an easily accessible model in which manipulation of bathing media on either the internal or external surface allowed for alteration of the ionic composition as well as addition of factors that mediate ion flux. Ussing’s short circuit current technique was devised to remove the contribution of the native membrane potential and quantify the net electrogenic flux as the current required to maintain a zero potential difference. With the now well-known technique, he provided credence for the idea of active Na⁺ transport (2) and suggested the existence of a Na⁺ pump driven by metabolic energy. It was not until 7 years later that Jens Skou demonstrated the sodium pump was actually an ATPase activated by both Na⁺ and K⁺ (3).

In 1958 Ussing, together with Valborg Koefoed-Johnsen, proposed a “two-membrane” model to explain the lack of correlation between the electrical potential difference and the external cation concentration. In this model, the outer surface of the frog skin was behaving as a sodium electrode, whereas the inner surface was acting as a potassium electrode (1, 4). Those of us who regard ourselves as epithelial transport physiologists may do well to consider how we might explain this paradox of ion fluxes if we did not take for granted the concept of separate apical and basolateral membranes with a different complement of transport proteins in each.

These paradigm-shifting concepts, coupled with the simplicity of the Ussing chamber physiological technique, opened the epithelial transport field to studies in any tissue that could be mounted as a sheet in a Ussing chamber or, in later experiments, could be cultured as a monolayer on permeable supports. In essence, the epithelium of interest is mounted in the center of a chamber with ports for voltage and current electrodes, and each side is bathed in oxygenated, temperature-controlled media. The potential difference across the tissue is clamped to zero, and the resulting short-circuit current (I_SC) is a measure of net electrogenic transepithelial ion flux. The original Ussing electrophysiological design is shown in Fig. 1A. Several commercial versions are now available, and current-voltage clamps monitor the changes in short-circuit current continuously while providing the ability to intermittently change the voltage clamp from zero to a preset holding potential. Using Ohm’s law, the change in the current during the nonzero holding potential can be used to calculate the transepithelial electrical resistance (TEER). The inverse of the TEER is the conductance and represents the epithelial permeability, a measure of both transcellular and paracellular flux (Fig. 2B).

Figure 1.

A: original diagram of the Ussing chamber set-up and the figure legend from Ussing and Zerahn’s manuscript in 1951 (2). The diagram is reprinted with permission from John Wiley and Sons. B: Ussing chamber set-up as applied to cells cultured on permeable supports. Figure created with BioRender.com by Alexandra Hochstetler. TEER, transepithelial electrical resistance.

Figure 2.

Current voltage clamp and double chamber Ussing chambers: current voltage clamp made by Gus Kelley and Gary Huber in the group of Professor Malcolm Cox at the University of Pennsylvania, Renal Electrolyte Section. A: the clamp shown was made to measure both short-circuit current and transepithelial resistance depending on the placement of the electrical connections. B: the picture shows a double chamber (made for toad urinary bladder) wherein the tissue to be measured was placed over both chambers. When the two halves were assembled, the rubber gaskets separated the tissue and provided two electrically isolated pieces. The white plugs on the sides had small ports for the insertion of agar bridges to connected to electrodes that interfaced with the clamp apparatus in A.

TOAD BLADDERS AND XENOPUS LAEVIS KIDNEY CELLS: A HISTORICAL PERSPECTIVE

Following in Prof. Ussing’s footsteps, my ion transport experiments as a graduate student in the Renal Electrolyte Section of the University of Pennsylvania School of Medicine used amphibian models—first the urinary bladder of the cane toad Bufo marinus and then one of the first continuous “renal” cell lines, the A6 line derived from the kidney of the South African clawed toad, Xenopus laevis. I studied hormonal regulation of Na⁺ transport using a voltage clamp apparatus that was built by members of the laboratory of Dr. Malcolm Cox (Fig. 2). This was at a time when core facilities consisted of a machine shop that could fashion equipment (in this case Ussing chambers) according to the investigator’s specifications. The Cox laboratory had designed a “double chamber” in which the tissue that was placed across the chamber could be isolated into two parts that could be monitored and manipulated separately (Fig. 2). These chambers were ideal for the large, freshly dissected urinary bladders from Bufo marinus. Since the cane toad has a bilobed urinary bladder, each relatively large hemibladder could be opened and placed across one double chamber, thereby providing four identical pieces of tissue from the same animal, which was invaluable for well-controlled experiments. Due to the unique renal-like absorptive characteristics of toad bladders, we were able to use this model to study the control of transepithelial Na⁺ transport in response to aldosterone, insulin, and insulin-like growth factor 1 (5–8). The advent of the A6 cell line meant that we no longer had to deal with the problems of importing animals internationally (toads from the Dominican Republic showed the most consistent responses). We found that responses to mammalian hormones in the A6 line were remarkably similar to those of the toad urinary bladder and, in later experiments, to mammalian renal cell lines (9–11).

The importance of Na⁺ flux into epithelial cells across the apical (pond-facing in frog skin) membrane was known but the definitive identity and cloning of the epithelial Na⁺ channel (ENaC) by Cecilia Canessa and Bernard Rossier (12, 13) provided the wherewithal for subsequent studies proving the physiological and medical importance of this channel. Interestingly it is ENaC, on the apical membrane, and not Na⁺K⁺ATPase on the basolateral membrane, that is regulated by hormones and other factors that control Na⁺ reabsorption whether in the frog skin or the principal cells of the distal nephron (4, 14; Fig. 3).

Figure 3.

Schematic of ENaC-mediated transepithelial electrolyte flux. ADH, antidiuretic hormone; ENaC, epithelial Na⁺ channel.

The excellent work of Canessa and Rossier demonstrated the evolutionary conservation of ENaC structure (13). Together with those of many other laboratories, our studies showed that there was also a remarkable conservation of the hormonal pathways that regulate this important ion channel. For example, with Ussing-style electrophysiological techniques, we showed that physiologically relevant concentrations of mammalian hormones (aldosterone, insulin, and insulin-like growth factor 1) stimulate remarkably similar electrogenic ion flux in toad urinary bladders, A6 cells, or mouse cortical collecting duct cell lines (14). I once heard Cecilia Canessa express the sentiment that almost everything we know about ENaC was first discovered in amphibian tissue and a reading of the ENaC literature from the 1980s and 1990s certainly substantiates that claim.

Given the importance of controlling electrolyte transport in both physiology and medicine, the development of mammalian models, including renal, pulmonary, and intestinal cell lines, revolutionized the study of not only ENaC but other electrogenic ion fluxes important in disease mechanisms underlying such important maladies as hypertension, cystic fibrosis, secretory diarrhea, and polycystic kidney disease (PKD) to mention a few. In the remainder of this article, I would like to discuss more recent studies applying Ussing chamber physiology to processes involved in the production of cerebrospinal fluid (CSF) by the choroid plexus epithelium (CPe).

FROM KIDNEY TO BRAIN

My interest in the choroid plexus arose from a serendipitous observation while exploring potential therapies for the treatment of PKD (15–17). Spurred by a discussion with Oleh Pochynyuk during a poster session at Experimental Biology, my colleague Vince Gattone and I agreed to look at the effect of a transient receptor potential vanilloid 4 (TRPV4) channel agonist on renal function in a PKD rodent model. TRPV4, a mechano-, osmo-sensitive channel was known to increase Ca²⁺ influx and, according to what is known about intracellular signaling in cystic disease, could be postulated to be effective in halting cyst growth. Due to financial constraints, we chose the rapidly progressing Tmem67 (formerly called Wpk) rat, which is orthologous to a rare human disease called Meckel–Gruber syndrome, type 3 (18, 19). The affected humans and animals carry a single point mutation in TMEM67, one of a complex of proteins involved in protein transport through the transition zone of the primary cilium. The homozygous affected animals have severe renal cystic disease and hydrocephalus and typically survive for only 18–21 days after birth (19).

In this rapidly progressing model of renal cystic disease, we were unable to show any ameliorating effects of TRPV4 agonists on cyst growth. It is important to note that in subsequent experiments in more relevant, slowly progressing models, Dr. Pochynyuk and his colleagues have been successful in elucidating the role of TRPV4 in the development of PKD (20, 21). In addition, this laboratory has subsequently shown that TRPV4 is important for sensing, and responding to, changes in flow and flow-dependent K⁺ secretion in the distal renal tubule (22).

During a TRPV4 agonist treatment regimen in the Tmem67 model, it was noted that the homozygous, severely affected animals seemed to exhibit a shortened lifespan when treated with the agonist. Interestingly, TRPV4 agonist treatment was found to have an adverse effect on the hydrocephalus in this model as measured by head dimensions in the very young animals. This, of course, begs the question of whether a TRPV4 antagonist would have the opposite effect. Indeed, using the more sophisticated technique of MRI, we were able to show that treatment with a TRPV4 antagonist arrested the progress of hydrocephalus in this model (23).

THE TRPV4 CHANNEL

To a cell physiologist, these findings raise interesting questions regarding the role of TRPV4 in CSF production. TRPV4 is found in the plasma membrane of epithelial, neural, and endothelial cells in a wide variety of organs (24–26). TRPV4 is expressed in both native choroid plexus epithelia (CPe) and in a CPe-derived cell line (27). The channel is reported to be activated by changes in osmotic balance (hypotonicity), mechanical stress (pressure), elevations in temperature, and inflammatory mediators such as arachidonic acid derivatives (24–26, 28–31). Considering the endogenous activators, TRPV4 can be regarded as a stress channel that is predominately activated under nonhomeostatic conditions. When activated, TRPV4 transports Ca²⁺, Na⁺, and other cations into cells, stimulating intracellular Ca²⁺ signaling processes including activation of Ca²⁺-sensitive channels such as those responsible for transepithelial ion fluxes (26) and can also change the transmembrane Na⁺ gradient.

In agreement with a role of TRPV4 as a stress-induced channel, it has been shown to be a crucial component of cytokine release during inflammatory responses (32, 33). In one of the first studies to show TRPV4 involvement in cytokine production by epithelial cells, D’Aldebert et al. (32) found that the addition of a TRPV4 agonist to intestinal epithelial cell lines resulted in the activation of proinflammatory signaling pathways and the production of interleukin (IL)-8, interferon γ-inducible protein (IP-10), monokine induced by interferon γ (MIG) and regulated on activation, normal T cell expressed and secreted (RANTES) . Subsequent experiments in human respiratory epithelial cell lines demonstrated similar results showing a TRPV4 agonist-induced increase in IL-8 (33).

One of the most interesting functional changes in response to TRPV4 activation is an increase in barrier cell permeability. In mammary epithelial cells in culture, TRPV4 activation caused a substantial decrease in transepithelial electrical resistance that was due to activation of a large conductance K⁺ channel and a slow increase in paracellular permeability accompanied by dramatic changes in tight junction morphology (34). In human retinal microvascular endothelial cells (HrMVECs) a TRPV4 agonist activated a nonselective cation current, elevated intracellular Ca²⁺, and reversibly increased the permeability of MVEC monolayers. The permeability change was associated with the disrupted organization of endothelial F-actin, downregulated expression of occludin, and remodeling of adherens contacts (35).

Several studies, including our own, have examined the effect of TRPV4 activation on choroid plexus epithelial cells in culture (27, 36–38). In primary CPe cultures, TRPV4 activation resulted in a change in TEER from ∼60 Ω·cm² to 20 Ω·cm² with a concurrent disintegration of the cell junctions (36). In accordance with the very low resistance, the images of the changes in the CPe cultures after TRPV4 activation indicate “holes” in the cellular monolayer, a scenario that is incompatible with the normal function of the CPe as a barrier separating the filtrate of a fenestrated capillary network from the CSF. Using both porcine and human continuous cell lines of the CPe, we have shown that TRPV4 activation is accompanied by a substantial decrease in TEER, which represents a corresponding increase in cellular permeability (27, 37, 38). Ussing-style electrophysiology was particularly useful in allowing us to follow changes in both TEER and I_SC over time and to show that the changes in response to low dose agonist were immediately reversible by the addition of a TRPV4 antagonist (Fig. 4). The lack of disintegration of the junctional complexes, suggested by the reversibility, was confirmed by immunocytochemistry. It is our working hypothesis, based on our own studies as well as the published literature, that the degree of junctional changes, and hence the integrity of the barrier, is dependent on the concentration of the agonist.

Figure 4.

Ussing-style electrophysiological experiments of TRPV4 agonist and antagonist in choroid plexus cell lines. The graphs represent a compilation of several studies. Electrophysiology graphs are displayed as I_SC (left, a measure of net electrogenic ion flux) and conductance (right, a measure of barrier permeability). GSK1016790A, a TRPV4 agonist, was added to all cultures at time zero using a concentration that was determined optimal for each cell line.A: electrophysiological responses in the porcine choroid plexus-Reims cell line. The black trace represents the agonist-only treatment. In the cultures shown by the pick traces, a TRPV4 antagonist, RN1734 was added 10 min before the addition of the agonist. The antagonist prevents the TRPV4-mediated increase in ion flux and conductance. In the teal trace, RN 1734 added bilaterally 10 min after the addition of agonist. The antagonist immediately reverses both the current and conductance.B: electrophysiological responses in a human choroid plexus cell line, HIBCCP. The black trace represents the agonist only treatment. In the cultures shown by the pick traces, a TRPV4 antagonist, RN1734 was added 5 min before the addition of the agonist. The antagonist prevents the TRPV4-mediated increase in ion flux and conductance. In the teal trace, RN 1734 added bilaterally at time t = 5 min. The antagonist immediately reverses both the current and conductance. All traces represent means ± SE for the stated “n” of technical replicates. *P < 0.05 considered significant between condition and GSK control measured by multiple t tests grouped analysis and indicated by color. GSK, GSK1016790A; HIBCPP, human choroid plexus papilloma; I_sc, short circuit current; TRPV4, transient receptor potential vanilloid 4.

Paradoxically, in cultured human epidermal keratinocytes, prolonged incubation (48–60 h) with a TRPV4 agonist strengthened the tight junction barrier and increased TEER (39). The differences between the epithelial and epidermal studies may reflect the timeframe of measurement. Most TEER measurements in epithelial cells are performed within minutes to hours after TRPV4 activation, not chronically as in the epidermal studies.

In vivo, the findings are consistent with a TRPV4-mediated increase in barrier permeability in multiple cell types. Intravenous administration of a TRPV4 agonist caused a dose-dependent reduction in blood pressure, followed by profound circulatory collapse in three species (mouse, rat, and dog). Hemodynamic analyses in the dog and rat demonstrated a substantial reduction in cardiac output that was associated with extensive vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. The authors concluded that activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier (40).

TRPV4 antagonists have been tested in clinical trials for a variety of indications. GlaxoSmithKline (GSK) completed several clinical trials using GSK2798745, an orally bioavailable TRPV4 channel blocker (ClinicalTrials.gov; search term TRPV4). In a 7-day trial in patients with congestive heart failure, the primary metric was changes in pulmonary gas transfer and respiration. An additional study used the same compound for chronic cough and alveolar barrier disruption in segmental lipopolysaccharide challenge. In these trials, the antagonist caused no severe adverse effects. A published report described a first-in-human study to evaluate safety, tolerability, pharmacodynamics, and pharmacokinetics of GSK2798745 in healthy subjects and stable patients with heart failure (41). Five cohorts contained a total of 60 participants who received the antagonist. Again, there was no mortality or serious drug-related adverse effects. GSK is currently recruiting for a study assessing treatment with the antagonist in diabetic macular edema. Combined, the clinical trials suggest that inhibiting TRPV4 will have minimal side effects. This was presaged by animal studies showing that Trpv4 null mice have a normal appearance, growth, and reproductive capacity and only develop osmotic abnormalities when placed under severe osmotic stress (31). All of the preclinical studies indicate that inhibition of TRPV4 as a clinical therapy will be safe.

EARLY STUDIES OF CHOROID PLEXUS EPITHELIA

The main tenants of transepithelial transport in the choroid plexus were established by Ernest Wright and colleagues in an extensive series of studies that spanned over a decade from 1972 to 1984 (reviewed in Ref. 42). This work used native choroid plexuses from the frog to establish the direction and control of the primary ion fluxes, the amount of CSF secretion, and the interesting and unique finding that Na⁺K⁺ATPase localized to the apical membrane. Following these elegant studies, many investigators contributed to filling in the myriad of transporters, and the reader is referred to excellent review articles by Jeppe Praetorius and Helle Damkier for the most current understanding of the major transporters and their localization (43, 44).

USSING-STYLE ELECTROPHYSIOLOGICAL STUDIES IN CHOROID PLEXUS EPITHELIAL CELLS

Due to difficulties in using freshly isolated CPe and wishing to confine our studies to mammalian epithelia, we have characterized the transport properties of two CPe cell lines, the porcine choroid plexus-Riems (PCP-R) and the human choroid plexus papilloma (HIBCPP) lines (45, 46). These lines were kindly provided by Horst Schroten and Christian Schwerk from Heidelberg University and were chosen because they exhibit polarized transport as well as a transepithelial electrical resistance indicative of a barrier epithelium. The latter property is relatively unique in cultured CPe and is crucial to Ussing electrophysiological studies.

Our initial studies were performed in the porcine choroid plexus-Riems (PCP-R) cell line, which is relatively easy to culture and is amenable to Ussing-style electrophysiology. Using this line, we showed that activation of TRPV4 with a specific agonist, GSK1016790A, resulted in an immediate increase in both transepithelial ion flux (I_SC) and conductance (the inverse of the TEER). The increase in ion flux was polymodal and was inhibited by either of two distinct antagonists, HC067047 or RN1734 (Fig. 4 shows the effect of RN1734). It was in these early experiments that we showed that the change in conductance was reversible and did not involve disruption of epithelial junctional complexes. To dissect the polymodal nature of the ion flux response, we reasoned that since TRPV4 activation results in an influx of Ca²⁺ into the cells, part of the response may be due to Ca²⁺-activated channels. PCP-R cells endogenously contain two types of Ca²⁺-activated K⁺ channels, the small conductance 2 (SK2) and the intermediate conductance (IK) channels. Based on inhibitor studies, the former is not involved in the TRPV4-mediated electrophysiological changes, whereas one of the three isoforms of the IK channel (KCNN4c) appears to play a role in the TRPV4 agonist-mediated electrogenic flux and permeability changes (27). The other aspects of the response likely involve Cl⁻ channels (D. Preston and B. Blazer-Yost, unpublished observations) but remain incompletely characterized.

In subsequent studies, we used this line to explore the finding that, in other tissues, TRPV4 is activated by cytokines and inflammatory mediators. We found that select proinflammatory cytokines [TNF-α, IL-1β, and transforming growth factor (TGF)-β1] had inhibitory effects on TRPV4-stimulated transepithelial ion flux and conductance changes, whereas anti-inflammatory cytokines (IL-10, IL-4, and IL-6) had no effect. Contrary to published studies, the proinflammatory mediator arachidonic acid (AA) had inhibitory rather than stimulatory effects on TRPV4-mediated responses. However, inhibition of AA metabolism also caused inhibitory effects on TRPV4, suggesting a complex interaction of AA and its metabolites in the regulation of TRPV4 activity (37). Although interesting, these results suggest that the role of the CPe in the inflammatory response may be more as a factor in regulating the production of cytokines and inflammatory mediators rather than reacting to them. These studies are ongoing.

Unfortunately for a variety of reasons, we began to suspect that at least some of the transporters and pumps present in the PCP-R cell line were mispolarized. In the first instance, in Ussing-style electrophysiological experiments, we noted that TRPV4 agonists were more effective when added to the basolateral bathing media while in vivo TRPV4 is located in the apical membrane. A more definitive proof of the mispolarization, however, came in the form of collaborative studies with Eric Delpire (38).

As demonstrated by multiple investigators, both the Na⁺ K⁺-ATPase pump and the NKCC1 cotransporter are localized on the apical membrane of native CPe and are involved in K⁺ influx into the cell (42, 46–50). However, in the PCP-R cells, when K⁺ influx was measured utilizing radioactive ⁸³Rb as a congener of K⁺, we discovered this may not be the case in these cells. Ouabain, an inhibitor of Na⁺K⁺ATPase, and bumetanide, an inhibitor of NKCC1, were used to measure inhibition of K⁺ influx from either the apical or basolateral compartment of the cultures. These studies indicated that significantly more K⁺ influx occurred across the basolateral membrane than the apical membrane in the PCP-R cells. Addition of both compounds resulted in an additive inhibition of 93% of K⁺ influx across the basolateral membrane. Therefore, in the PCP-R cell line, unlike in the native CPe, these transporters are localized and function primarily on the basolateral membrane. As a result, interpretations regarding fluid/electrolyte flux and the resultant CSF production using the PCP-R cell line should be pursued with caution (38). Although this line is certainly useful for studies examining factors, including infectious agents, that cause changes in junctional permeability, it is of limited use in studies directed toward characterizing the factors controlling the production and composition of CSF.

More recently, Alexandra Hochstetler and Louise Hulme in my laboratory have determined the optimal culture conditions for growing the human choroid plexus papilloma (HIBCPP) cell line and determined that this line is correctly polarized at least with respect to Na⁺K⁺ATPase, TRPV4, NKCC1, and anion exchange protein 2 (AE2). Activation of the endogenous TRPV4 also results in a multiphasic transepithelial ion flux albeit with an opposite directionality when compared with the PCP-R cell line (Fig. 4). The TRPV4 agonist-induced electrolyte flux is sensitive to TRPV4 antagonists as well as inhibitors of the intermediate conductance Ca²⁺ sensitive K⁺ channel (IK). The Ca²⁺-activated Cl⁻ channel TMEM16a (ANO1) may also play a role in the electrogenic flux (51 and B. Blazer-Yost, unpublished data).

One of the most intriguing parts of the response in either cell line is the substantial change in permeability (measured as conductance), the reversibility of which is dose dependent. At low nanomolar concentrations of the widely used agonist GSK1016790A, the change in permeability is immediately reversible with specific antagonists such as RN 1734 or HC067047, whereas at higher agonist concentrations, the permeability change climbs to an irreversible level that ultimately disturbs the monolayer, opening “holes” in the monolayer structure.

TRPV4 is highly expressed in both the native epithelium and in the CPe cell lines (23, 24). It is important to note that the endogenous activation of TRPV4 by pressure, osmotic changes, or arachidonic acid derivatives is rarely used in vitro or in preclinical animal studies. In many cases, the in vivo agonists are only postulated. However, it is logical to assume that physiological activation of TRPV4, even under stress conditions, is likely to fall within the reversible range of permeability changes. Therefore, care should be exercised when activating TRPV4 with exogenous chemicals rather than endogenous stimulatory agents. To mimic the in vivo situation, it is necessary to select a concentration that is likely to have physiological, nontoxic effects.

The magnitude of the change in resistance/permeability within the reversible range is something that is not, to my knowledge, found in most other epithelial cell types and cultured cell lines. In fact, the level of permeability change assessed by Ussing chamber electrophysiology in the choroid plexus cell lines is shared predominately with intestinal epithelial cells that have been stimulated to mimic secretory diarrhea. Dan Halm has characterized secretory processes in the distal colon from various species (e.g., 52, 53) and has divided these secretory mechanisms into three distinct phases, flushing secretion, modulatory secretion, and synergistic secretion based on the rate and duration of the K⁺ and Cl⁻ secretory processes as well as the secretagogues, which stimulate the response (52).

The comparison of the substantial permeability changes between the distal colon and the choroid plexus is fascinating, however, Ussing style electrophysiological studies have also revealed differences between these two tissues. Although the changes in conductance are likely due to K⁺ and Cl⁻ secretion in both tissues, the control of the driving forces is different. Electrogenic K⁺ secretion in the colon is completely sensitive to bumetanide suggesting a requirement for Na⁺K⁺Cl⁻ cotransporters (53). In choroid plexus epithelial cell lines, our studies indicate that bumetanide has no effect on either basal or TRPV4-stimulated electrogenic ion flux (B. Blazer-Yost, unpublished data). In animal models, NKCC1 is thought to play a role in CSF production in some conditions but not others (e.g., 54, 55). With regard to the choroid plexus per se, the controversial aspects of the exact role of NKCC1 are ongoing as evidenced by a recent CrossTalk series in Journal of Physiology (56–59). The controversial aspects of these issues are beyond the scope of this article, but it is likely that Ussing type electrophysiological studies will be involved in many of the future studies.

ONGOING STUDIES

Our studies on the unique epithelium of the choroid plexus are ongoing and Prof Ussing’s legacy is very much a part of these studies. We are currently focused on the regulation of the production of CSF and how TRPV4 contributes to this process in health and disease. CPe do not have ENaC and yet active transport dictates an increase in Na⁺ flux to drive NaK⁺ATPase. An interesting question is what is the Na⁺ influx pathway and how does TRPV4 influence that process? Although the cation influx commonly associated with TRPV4 activation is Ca²⁺, the channel also transports Na⁺ and we have shown that activation of TRPV4 results in an immediate and substantial increase in Na⁺ influx in both native, freshly isolated rat CPe and in the PCP-R cell line (23 and B. Blazer-Yost, unpublished data). The importance of this influx in contributing to active transport as well as the physiologically relevant conditions under which it occurs remain unknown.

Another interesting aspect of these studies is the question of what regulates TRPV4 in vivo and whether it is a “maintenance channel” or a “stress channel.” What are the intracellular signaling pathways controlling TRPV4 activity? Ussing style electrophysiological studies are very well suited for such investigations.

An ongoing controversy between multiple investigators is the role of NKCC1 in the CPe and under what conditions this transporter plays a role in CSF production. The net ion flux after NKCC1 stimulation is electroneutral and, therefore, not detectable by Ussing-style electrophysiology. However, if this channel is an important component of CSF production, one would assume that inhibiting it would alter other, electrogenic, fluxes. We have not detected evidence of such activity in the CPe cell lines and are currently designing experiments to examine the role of NKCC1 in fluid secretion by the cell culture models.

All of these questions are aspects of an overall aim of determining the transepithelial fluxes stimulated by TRPV4 that may contribute to the formation and composition of the CSF. Unlike renal principal cells, which are “well behaved” and ultimately show a transepithelial flux that is predominately due to Na⁺ absorption, the I_SC response of the CPe contains multiple components. We are in the early stages of dissecting which of these are primary and whether there are bidirectional fluxes with different temporal signatures.

GRANTS

The most recent work was supported by a Hydrocephalus Association Innovator award and by the U.S. Department of Defense Grant W81XWH-16-PRMRP-IIRA.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

B.L.B.-Y. prepared figures; drafted manuscript; edited and revised manuscript; approved final version of manuscript.