Molecular water pumps.

There is good evidence that cotransporters of the symport type behave as molecular water pumps, in which a water flux is coupled to the substrate fluxes. The free energy stored in the substrate gradients is utilized, by a mechanism within the protein, for the transport of water. Accordingly, the water flux is secondary active and can proceed uphill against the water chemical potential difference. The effect has been recognized in all symports studied so far (Table 1). It has been studied in details for the K+/Cl- cotransporter in the choroid plexus epithelium, the H+/lactate cotransporter in the retinal pigment epithelium, the intestinal Na+/glucose cotransporter (SGLT1) and the renal Na+/dicarboxylate cotransporter both expressed in Xenopus oocytes. The generality of the phenomenon among symports with widely different primary structures suggests that the property of molecular water pumps derives from a pattern of conformational changes common for this type of membrane proteins. Most of the data on molecular water pumps are derived from fluxes initiated by rapid changes in the composition of the external solution. There was no experimental evidence for unstirred layers in such experiments, in accordance with theoretical evaluations. Even the experimental introduction of unstirred layers did not lead to any measurable water fluxes. The majority of the experimental data supports a molecular model where water is cotransported: A well defined number of water molecules act as a substrate on equal footing with the non-aqueous substrates. The ratio of any two of the fluxes is constant, given by the properties of the protein, and is independent of the driving forces or other external parameters. The detailed mechanism behind the molecular water pumps is as yet unknown. It is, however, possible to combine well established phenomena for enzymes into a working model. For example, uptake and release of water is associated with conformational changes during enzymatic action; a specific sequence of allosteric conformations in a membrane bound enzyme would give rise to vectorial transport of water across the membrane. In addition to their recognized functions, cotransporters have the additional property of water channels. Compared to aquaporins, the unitary water permeability is about two orders of magnitude lower. It is suggested that the water permeability is determined from chemical associations between the water molecule and sites within the pore, probably in the form of hydrogen-bonds. The existence of a passive water permeability suggests an alternative model for the molecular water pump: The water flux couples to the flux of non-aqueous substrates in a hyperosmolar compartment within the protein. Molecular water pumps allow cellular water homeostasis to be viewed as a balance between pumps and leaks. This enables cells to maintain their intracellular osmolarity despite external variations. Molecular water pumps could be relevant for a wide range of physiological functions, from volume regulation in contractile vacuoles in amoeba to phloem transport in plants (Zeuthen 1992, 1996). They could be important building blocks in a general model for vectorial water transport across epithelia. A simplified model of a leaky epithelium incorporating K+/Cl-/H2O and Na+/glucose/H2O cotransport in combination with channels and primary active transport gives good quantitative predictions of several properties. In particular of how epithelial cell layers can transport water uphill.

An electrogenic NA+/K+ pump in the choroid plexus.

Intracellular electrical potential and potassium activity was measured by means of microelectrodes in the epithelial cells of choroid plexus from bullfrogs (Rana catesbeiana). Ouabain applied from the ventricular side caused an abrupt depolarisation of 10 mV but only a gradual loss of potassium from the cells. Readministration of potassium to the ventricular solution of plexuses which were previously depleted of potassium, caused a hyperpolarisation of about 4 mV. These two experiments are consistent with the notion of an electrogenic Na+/K+ pump situated at the ventricular membrane and which pumps potassium into the cell and sodium into the ventricle. The numerical values obtained suggest that 3 sodium ions are pumped for 2 potassium ions. The permeability coefficient for potassium exit from the cell is calculated to be 1.24 . 10(-5) cm-1 . s-1 expressed per cm2 of flat epithelium.

ATPase activity of P-glycoprotein related to emergence of drug resistance in Ehrlich ascites tumor cell lines.

We have characterized the ATPase activity of a sensitive and five progressively daunorubicin resistant Ehrlich ascites tumor cell lines passaged in mice. For the nine different modulators of drug resistance that we have studied, the ATPase activity first rose with the modulator concentration and then declined. We analyzed the ATPase activity profiles in terms of an activation constant and an inhibition constant for each of the nine drugs and six cell lines. In this series of cell lines, the drug-stimulatable ATPase activity was directly proportional to the amount of P-glycoprotein. Pumping of daunorubicin was also correlated with the amount of P-glycoprotein, except that, for a highly passaged line more daunorubicin was pumped than could be accounted for by the content of P-glycoprotein. Between the 12th and the 36th passage an additional source of resistance emerged, which was not correlated with P-glycoprotein. Pumping of daunorubicin was negatively correlated with the cell volume for the different lines.

Structure-activity relationships of P-glycoprotein interacting drugs: kinetic characterization of their effects on ATPase activity.

We have determined the kinetic parameters for stimulation and inhibition by 34 drugs of the P-glycoprotein ATPase in membranes derived from CR1R12 Chinese hamster ovary cells. The drugs chosen were sets of calmodulin antagonists, steroids, hydrophobic cations, hydrophobic peptides, chemotherapeutic substrates of P-glycoprotein, and some other drugs with lower affinity for P-glycoprotein. We studied how these kinetic parameters correlated with the partition coefficient and the Van der Waals surface area of the drugs. The maximum velocity of ATPase stimulation decreased with surface area and showed a suggestion of a maximum with increasing partition coefficient. The affinity of these drugs for P-glycoprotein showed no significant correlation with partition coefficient but was highly correlated with the surface area suggesting that binding between modulators and P-glycoprotein takes place across a wide interaction surface on the protein.

Competitive, non-competitive and cooperative interactions between substrates of P-glycoprotein as measured by its ATPase activity.

We have studied the interaction between verapamil and other modulators of the P-glycoprotein ATPase from membranes of CR1R12 Chinese hamster ovary cells. Four major categories of interaction were identified. (i) Non-competitive inhibition of verapamil's stimulation of enzyme activity was found with vanadate. (ii) Competitive inhibition of the ATPase was found for the pair verapamil and cyclosporin A. (iii) Allosteric inhibition with an increase in the Hill number for verapamil was found in the cases of daunorubicin, epirubicin, gramicidin S and D, vinblastine, amiodarone, and colchicine. (iv) Cooperative stimulation of verapamil-induced ATPase activity was found with progesterone, diltiazem, amitriptyline, and propranolol. At high levels, progesterone and verapamil mutually enhanced each other's inhibitory action on the ATPase. Our data show that the substrate binding behavior of P-glycoprotein is complex with more than one binding site being present. This information could form the basis for the development of improved modulators of P-glycoprotein.

Molecular mechanisms for passive and active transport of water.

Water crosses cell membranes by passive transport and by secondary active cotransport along with ions. While the first concept is well established, the second is new. The two modes of transport allow cellular H2O homeostasis to be viewed as a balance between H2O leaks and H2O pumps. Consequently, cells can be hyperosmolar relative to their surroundings during steady states. Under physiological conditions, cells from leaky epithelia may be hyperosmolar by roughly 5 mosm liter-1, under dilute conditions, hyperosmolarities up to 40 mosm liter-1 have been recorded. Most intracellular H2O is free to serve as solvent for small inorganic ions. The mechanism of transport across the membrane depends on how H2O interacts with the proteinaceous or lipoid pathways. Osmotic transport of H2O through specific H2O channels such as CHIP 28 is hydraulic if the pore is impermeable to the solute and diffusive if the pore is permeable. Cotransport of ions and H2O can be a result of conformational changes in proteins, which in addition to ion transport also translocate H2O bound to or occlude in the protein. A cellular model of a leaky epithelium based on H2O leaks and H2O pumps quantitatively predicts a number of so-far unexplained observations of H2O transport.

Water transport in the brain: role of cotransporters.

It is generally accepted that cotransporters transport water in addition to their normal substrates, although the precise mechanism is debated; both active and passive modes of transport have been suggested. The magnitude of the water flux mediated by cotransporters may well be significant: both the number of cotransporters per cell and the unit water permeability are high. For example, the Na(+)-glutamate cotransporter (EAAT1) has a unit water permeability one tenth of that of aquaporin (AQP) 1. Cotransporters are widely distributed in the brain and participate in several vital functions: inorganic ions are transported by K(+)-Cl(-) and Na(+)-K(+)-Cl(-) cotransporters, neurotransmitters are reabsorbed from the synaptic cleft by Na(+)-dependent cotransporters located on glial cells and neurons, and metabolites such as lactate are removed from the extracellular space by means of H(+)-lactate cotransporters. We have previously determined water transport capacities for these cotransporters in model systems (Xenopus oocytes, cell cultures, and in vitro preparations), and will discuss their role in water homeostasis of the astroglial cell under both normo- and pathophysiologal situations. Astroglia is a polarized cell with EAAT localized at the end facing the neuropil while the end abutting the circulation is rich in AQP4. The water transport properties of EAAT suggest a new model for volume homeostasis of the extracellular space during neural activity.

Evaluation of a needle pH electrode for continuous tissue-pH monitoring during labor. Characteristics during acidosis in the rat.

A prototype of a needle tissue-pH (t-pH) electrode designed for continuous t-pH monitoring of the human fetus during labor is described and evaluated in vitro and in vivo in the rat. Respiratory acidosis was induced by ventilation with 5% carbon dioxide, and t-pH measured by the needle electrode compared to simultaneous t-pH measured by the Kontron-Roche electrode and to arterial blood pH. A close correlation between the two t-pH electrodes was demonstrated (r = 0.80, P < 0.001). Furthermore, a close correlation was found between t-pH and arterial blood pH during the first 15 min of acidosis (r = 0.82, P< 0.001). It is concluded that the needle electrode fulfils nearly all theoretical demands to a t-pH electrode for clinical use.

Local impermeant anions establish the neuronal chloride concentration.

Neuronal intracellular chloride concentration [Cl(-)](i) is an important determinant of γ-aminobutyric acid type A (GABA(A)) receptor (GABA(A)R)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCCs) move Cl(-) across the membrane, but accumulating evidence suggests factors other than the bulk concentrations of transported ions determine [Cl(-)](i). Measurement of [Cl(-)](i) in murine brain slice preparations expressing the transgenic fluorophore Clomeleon demonstrated that cytoplasmic impermeant anions ([A](i)) and polyanionic extracellular matrix glycoproteins ([A](o)) constrain the local [Cl(-)]. CCC inhibition had modest effects on [Cl(-)](i) and neuronal volume, but substantial changes were produced by alterations of the balance between [A](i) and [A](o). Therefore, CCCs are important elements of Cl(-) homeostasis, but local impermeant anions determine the homeostatic set point for [Cl(-)], and hence, neuronal volume and the polarity of local GABA(A)R signaling.

Response to comments on "Local impermeant anions establish the neuronal chloride concentration".

We appreciate the interest in our paper and the opportunity to clarify theoretical and technical aspects describing the influence of Donnan equilibria on neuronal chloride ion (Cl(-)) distributions.

Water transport between CNS compartments: contributions of aquaporins and cotransporters.

Large water fluxes continuously take place between the different compartments of the brain as well as between the brain parenchyma and the blood or cerebrospinal fluid. This water flux is tightly regulated but may be disturbed under pathological conditions that lead to brain edema formation or hydrocephalus. The molecular pathways by which water molecules cross the cell membranes of the brain are not well-understood, although the discovery of aquaporin 4 (AQP4) in the brain improved our understanding of some of these transport processes, particularly under pathological conditions. In the present review we introduce another family of transport proteins as water transporters, namely the cotransporters and the glucose uniport GLUT1. In direct contrast to the aquaporins, these proteins have an inherent ability to transport water against an osmotic gradient. Some of them may also function as water pores in analogy to the aquaporins. The putative role of cotransport proteins and uniports for the water flux into the glial cells, through the choroid plexus and across the endothelial cells of the blood-brain-barrier will be discussed and compared to the contribution of the aquaporins.

Vasopressin-dependent short-term regulation of aquaporin 4 expressed in Xenopus oocytes.

Aquaporin 4 (AQP4) is abundantly expressed in the perivascular glial endfeet in the central nervous system (CNS), where it is involved in the exchange of fluids between blood and brain. At this location, AQP4 contributes to the formation and/or the absorption of the brain edema that may arise following pathologies such as brain injuries, brain tumours, and cerebral ischemia. As vasopressin and its G-protein-coupled receptor (V1(a)R) have been shown to affect the outcome of brain edema, we have investigated the regulatory interaction between AQP4 and V1(a)R by heterologous expression in Xenopus laevis oocytes. The water permeability of AQP4/V1(a)R-expressing oocytes was reduced in a vasopressin-dependent manner, as a result of V1(a)R-dependent internalization of AQP4. Vasopressin-dependent internalization was not observed in AQP9/V1(a)R-expressing oocytes. The regulatory interaction between AQP4 and V1(a)R involves protein kinase C (PKC) activation and is reduced upon mutation of Ser(180) on AQP4 to an alanine. Thus, the present study demonstrates at the molecular level a functional link between the vasopressin receptor V1(a)R and AQP4. This functional interaction between AQP4 and V1(a)R may prove to be a potential therapeutic target in the prevention and treatment of brain edema.

Water permeability of ventricular cell membrane in choroid plexus epithelium from Necturus maculosus.

1. The osmotic water permeability Lp and the relations between the flows of H2O, K+ and Cl- were studied in the ventricular membrane of the epithelium from the choroid plexus of Necturus maculosus. 2. The flows were induced by abrupt changes in external osmolarity of the ventricular solution. Solution changes were convective and no effects of unstirred layers could be detected on measured parameters. 3. The initial rate of change in intracellular concentrations of K+ and Cl- was monitored by double-barrelled ion-selective microelectrodes. 4. The initial rate of flux of H2O could be monitored as the changes in the concentration of intracellular choline ions (Ch+i). When 0.5 mmol l-1 of choline chloride was added to the external solutions, Ch+i attained values of 1-5 mmol l-1. The dilution or concentration of Ch+i could be recorded by K+ electrodes since the sensitivity of these to Ch+ is more than 50 times greater than to K+. 5. The Lp of the ventricular membrane of the epithelium was 1.4-2.1 x 10(-4) cm s-1 (osmol l-1)-1 and independent of the direction of the induced water flux. Lp was unchanged in tissues adapted to osmolarities of half the physiological value. 6. The efflux of H2O induced by mannitol was associated with an instantaneous efflux of K+ which was inhibited by furosemide. The fluxes had a ratio of 40 mmol l-1. The influx of H2O induced by the removal of NaCl from the ventricular solution was associated with an instantaneous influx of K+. The H2O influx had a ratio to the flux of K+ of 70 mmol l-1. 7. The efflux of H2O induced by mannitol was associated with an efflux of Cl- which was inhibited by furosemide. The ratio of the two fluxes was in the range 15-44 mmol l-1. 8. The conclusion is that the Ch+ method gives a reliable measure of the movement of H2O across the ventricular membrane. The magnitude of the Lp and its relevance to transepithelial transport are discussed. The osmotically induced H2O movement is accompanied by furosemide-sensitive fluxes of K+ and Cl- of the same magnitude. This suggests that co-transport between H2O and KCl can take place in the membrane.