A. E. Hill

What are aquaporins for

The prime function of aquaporins (AQPs) is generally believed to be that of increasing water flow rates across membranes by raising their osmotic or hydraulic permeability. In addition, this applies to other small solutes of physiological importance. Notable applications of this ‘simple permeability hypothesis’ (SPH) have been epithelial fluid transport in animals, water exchanges associated with transpiration, growth and stress in plants, and osmoregulation in microbes. We first analyze the need for such increased permeabilities and conclude that in a range of situations at the cellular, subcellular and tissue levels the SPH cannot satisfactorily account for the presence of AQPs. The analysis includes an examination of the effects of the genetic elimination or reduction of AQPs (knockouts, antisense transgenics and null mutants). These either have no effect, or a partial effect that is difficult to explain, and we argue that they do not support the hypothesis beyond showing that AQPs are involved in the process under examination. We assume that since AQPs are ubiquitous, they must have an important function and suggest that this is the detection of osmotic and turgor pressure gradients. A mechanistic model is proposed—in terms of monomer structure and changes in the tetrameric configuration of AQPs in the membrane—for how AQPs might function as sensors. Sensors then signal within the cell to control diverse processes, probably as part of feedback loops. Finally, we examine how AQPs as sensors may serve animal, plant and microbial cells and show that this sensor hypothesis can provide an explanation of many basic processes in which AQPs are already implicated. Aquaporins are molecules in search of a function; osmotic and turgor sensors are functions in search of a molecule.

AQP and the Control of Fluid Transport in a Salivary Gland

Experiments were performed with the perfused rat submandibular gland in vitro to investigate the nature of the coupling between transported salt and water by varying the osmolarity of the source bath and observing the changes in secretory volume flow. Glands were submitted to hypertonic step changes by changing the saline perfusate to one containing different levels of sucrose. The flow rate responded by falling to a lower value, establishing a new steady-state flow. The rate changes did not correspond to those expected from a system in which fluid production is due to simple osmotic equilibration, but were much larger. The changes were fitted to a model in which fluid production is largely paracellular, the rate of which is controlled by an osmosensor system in the basal membrane. The same experiments were done with glands from rats that had been bred to have very low levels of AQP5 (the principal aquaporin of the salivary acinar cell) in which little AQP5 is expressed at the basal membrane. In these rats, salivary secretion rates after hypertonic challenges were small and best modelled by simple osmotic equilibration. In rats which had intermediate AQP5 levels the changes in flow rate were similar to those of normal rats although their AQP5 levels were reduced.Finally, perfused normal glands were subject to retrograde ductal injection of salines containing different levels of Hg2+ ions (0, 10 and 100 μM) which would act as inhibitors of AQP5 at the apical acinar membrane. The overall flow rates were progressively diminished with rising Hg2+ concentration, but after hypertonic challenge the changes in flow rates were unchanged and similar to those of normal rats.All these results are difficult to explain by a cellular osmotic model but can be explained by a model in which paracellular flow is controlled by an osmosensor (presumably AQP5) present on the basal membrane.

Paracellular fluid transport by epithelia.

The evidence that a major fraction of water crosses the paracellular route during isotonic fluid transfer is reviewed together with a description of the theory and experimental results derived from extracellular probe studies. Four transporting epithelia which have been studied using the method are gallbladder, intestine, Malpighian tubule, and salivary gland. It is concluded that paracellular probe flows are not due to simple convection generated by osmotic flow through the junctions but are generated by active fluid transport within the junction: a mechano-osmotic process. The geometry of the pathway involved would indicate that some salt accompanies the paracellular fluid, representing a hypo-osmotic flow. Transport of salt by the cell route, which may be accompanied by some water, represents a hypertonic flow. The problem then becomes one of balancing the two to produce an isotonic transportate. We suggest, using recent data from knockout mice, that some aquaporins are functioning in different epithelial tissues as osmo-comparators within a feedback loop that regulates the paracellular fluid flow rate. This results in an overall quasi-isotonic transport by the epithelium. The model is applied to forward-facing systems such as proximal tubule and backward-facing systems such as exocrine glands.

An Osmotic Model of the Growing Pollen Tube

Pollen tube growth is central to the sexual reproduction of plants and is a longstanding model for cellular tip growth. For rapid tip growth, cell wall deposition and hardening must balance the rate of osmotic water uptake, and this involves the control of turgor pressure. Pressure contributes directly to both the driving force for water entry and tip expansion causing thinning of wall material. Understanding tip growth requires an analysis of the coordination of these processes and their regulation. Here we develop a quantitative physiological model which includes water entry by osmosis, the incorporation of cell wall material and the spreading of that material as a film at the tip. Parameters of the model have been determined from the literature and from measurements, by light, confocal and electron microscopy, together with results from experiments made on dye entry and plasmolysis in Lilium longiflorum. The model yields values of variables such as osmotic and turgor pressure, growth rates and wall thickness. The model and its predictive capacity were tested by comparing programmed simulations with experimental observations following perturbations of the growth medium. The model explains the role of turgor pressure and its observed constancy during oscillations; the stability of wall thickness under different conditions, without which the cell would burst; and some surprising properties such as the need for restricting osmotic permeability to a constant area near the tip, which was experimentally confirmed. To achieve both constancy of pressure and wall thickness under the range of conditions observed in steady-state growth the model reveals the need for a sensor that detects the driving potential for water entry and controls the deposition rate of wall material at the tip.

The paracellular component of water flow in the rat submandibular salivary gland

1 The pathway of water flow during salivary secretion by the isolated, perfused rat submandibular gland was examined using a family of homologous radiodextran molecules as probes of paracellular fluid transfer. 2 The secretion/perfusate ratio (S/P) of the secreted probes versus molecular radius during fluid secretion evoked by ACh could be resolved into two components: one that fitted a free‐diffusion (Stokes‐Einstein) curve and indicated diffusion through large channels, and a convective component that was linearly related to radius. 3 The convective component had a cut‐off point at 0.5 nm (5 Å) radius and an S/P intercept of near 1.0 at the radius of water, which indicates that most of the volume flow was paracellular. 4 The nature of such a paracellular flow is discussed together with the possible integration of this volume flow with the cellular transport of ions, resulting in an isotonic primary secretion from the gland.

Fluid Transport: A Guide for the Perplexed

Readers of the physiological literature may be excused if they feel that fluid transport has become a complex and confusing field that is difficult to understand and to assess. The major theories of fluid-transporting epithelia are examined here with respect to their ability to explain quasi-isotonic fluid transport and its modulation by salt transport, osmotic permeability and basal tonicity. The basics of each theory are set out concisely, and their pros and cons are made explicit. Finally, a comparison is made in table form indicating their overall performance in relation to the problems of this difficult but important field.

A new approach to epithelial isotonic fluid transport : An osmosensor feedback model

A model for control of the transport rate and osmolarity of epithelial fluid (isotonic transport) is presented by using an analogy with the control of temperature and flow rate in a shower. The model brings recent findings and theory concerning the role of aquaporins in epithelia together with measurements of epithelial paracellular flow into a single scheme. It is not based upon osmotic equilibration across the epithelium but rather on the function of aquaporins as osmotic sensors that control the tonicity of the transported fluid by mixing cellular and paracellular flows, which may be regarded individually as hyper- and hypo-tonic fluids, to achieve near-isotonicity. The system is built on a simple feedback loop and the quasi-isotonic behavior is robust to the precise values of most parameters. Although the two flows are separate, the overall fluid transport rate is governed by the rate of salt pumping through the cell. The model explains many things: how cell pumping and paracellular flow can be coupled via control of the tight junctions; how osmolarity is controlled without depending upon the precise magnitude of membrane osmotic permeability; and why many epithelia have different aquaporins at the two membranes.The model reproduces all the salient features of epithelial fluid transport seen over many years but also indicates novel behavior that may provide a subject for future research and serve to distinguish it from other schemes such as simple osmotic equilibration. Isotonic transport is freed from constraints due to limited permeability of the membranes and the precise geometry of the system. It achieves near-isotonicity in epithelia in which partial water transport by co-transporters may be present, and shows apparent electro-osmotic effects. The model has been developed with a minimum of parameters, some of which require measurement, but the model is flexible enough for the basic idea to be extended both to complex systems of water and salt transport that still await a clear explanation, such as intestine and airway, and to systems that may lack aquaporins or use other sensors.

The Limonium Salt Gland: A Biophysical and Structural Study

Publisher Summary This chapter discusses the structure of Limonium salt gland. The Limonium gland is a multicellular structure embedded in the leaf surface approximately 30 x 1 -6 -meter in diameter, comprising 16 small cells enclosed in a cutinized envelope; there are about 100 per square centimeter. Limonium salt glands confer euryhalinity upon a plant, which can tolerate an enormous range of salinity from fresh water to double seawater and beyond. This is of great importance to an organism that finds its ecological niche in estuarine marshes where the saline gradient is very steep and is changing with time at any point. The salt gland cells are able to cope with this situation by regulating their rates of ion transport to suit the prevailing salt load to which they are subjected. Thus, the salt gland cell is a highly specialized plant cell capable of providing answers to some of the most fundamental questions about the synthesis and functioning of membrane transport systems.

Osmotic Flow in Membrane Pores

A comparison is made between the conventional macroscopic pore theory, the single-file (no-pass) theory, and the bimodal theory in their ability to predict the values of the unit osmotic permeability P os of single pores. In larger pores osmosis is thought to be a viscous (bulk) flow, while in molecular-sized pores only diffusive flow is considered possible. The physical assumptions underlying these theories are examined and compared with bimodal theory in which (i) viscous flow is impossible in any pore region that can be permeated by the driving osmolyte, and (ii) a distinction between diffusive and viscous flow can still be present in no-pass pores. Experimental values for the osmotic permeability of channels formed by the antibiotics amphotericin, nystatin, and gramicidin and the cellular aquaporin CHIP28 determined with different osmolytes are compared with theoretical expressions for P os as a function of osmolyte radius. Aquaporins are probably pores of variable internal cross section and bimodal theory predicts that they can be probed by osmolytes of different radius to give different osmotic flows, although the overall permeability to each molecule is apparently zero. Such information can be used to construct a model of the pore channel. Conversely, if the pore structure is known, the unit osmotic permeability to any osmolyte can be calculated.

Mercury-sensitive water channels as possible sensors of water potentials in pollen

The growing pollen tube is central to plant reproduction and is a long-standing model for cellular tip growth in biology. Rapid osmotically driven growth is maintained under variable conditions, which requires osmosensing and regulation. This study explores the mechanism of water entry and the potential role of osmosensory regulation in maintaining pollen growth. The osmotic permeability of the plasmalemma of Lilium pollen tubes was measured from plasmolysis rates to be 1.32±0.31×10–3 cm s–1. Mercuric ions reduce this permeability by 65%. Simulations using an osmotic model of pollen tube growth predict that an osmosensor at the cell membrane controls pectin deposition at the cell tip; inhibiting the sensor is predicted to cause tip bursting due to cell wall thinning. It was found that adding mercury to growing pollen tubes caused such a bursting of the tips. The model indicates that lowering the osmotic permeability per se does not lead to bursting but rather to thickening of the tip. The time course of induced bursting showed no time lag and was independent of mercury concentration, compatible with a surface site of action. The submaximal bursting response to intermediate mercuric ion concentration was independent of the concentration of calcium ions, showing that bursting is not due to a competitive inhibition of calcium binding or entry. Bursting with the same time course was also shown by cells growing on potassium-free media, indicating that potassium channels (implicated in mechanosensing) are not involved in the bursting response. The possible involvement of mercury-sensitive water channels as osmosensors and current knowledge of these in pollen cells are discussed.