A Cl 2 Cotransporter Selective for NH 4 1 Over K 1 in Glial Cells of Bee Retina

There appears to be a flux of ammonium (NH 4 1 /NH 3 ) from neurons to glial cells in most nervous tissues. In bee retinal glial cells, NH 4 1 /NH 3 uptake is at least partly by chloride-dependant transport of the ionic form NH 4 1 . Transmembrane transport of NH 4 1 has been described previously on transporters on which NH 4 1 replaces K 1 , or, more rarely, Na 1 or H 1 , but no transport system in animal cells has been shown to be selective for NH 4 1 over these other ions. To see if the NH 4 1 -Cl 2 cotransporter on bee retinal glial cells is selective for NH 4 1 over K 1 we measured ammonium-induced changes in intracellular pH (pH i ) in isolated bundles of glial cells using a fluorescent indicator. These changes in pH i result from transmembrane fluxes not only of NH 4 1 , but also of NH 3 . To estimate transmembrane fluxes of NH 4 1 , it was necessary to measure several parameters. Intracellular pH buffering power was found to be 12 mM. Regulatory mechanisms tended to restore intracellular [H 1 ] after its displacement with a time constant of 3 min. Membrane permeability to NH 3 was 13 m m s 2 1 . A numerical model was used to deduce the NH 4 1 flux through the transporter that would account for the pH i changes induced by a 30-s application of ammonium. This flux saturated with increasing [NH 4 1 ] o ; the relation was fitted with a MichaelisMenten equation with K m < 7 mM. The inhibition of NH 4 1 flux by extracellular K 1 appeared to be competitive, with an apparent K i of z 15 mM. A simple standard model of the transport process satisfactorily described the pH i changes caused by various experimental manipulations when the transporter bound NH 4 1 with greater affinity than K 1 . We conclude that this transporter is functionally selective for NH 4 1 over K 1 and that the transporter molecule probably has a greater affinity for NH 4 1 than for K 1 . key words: ammonia • K-Cl cotransporter • neuroglia • pH • Apis I N T R O D U C T I O N Although transmembrane transport of ammonium in animals has been studied, mainly in the mammalian kidney, there are two well-established cases of fluxes of ammonium from neurons to glial cells in nervous tissue. In vertebrate brain, where glutamate is the main neurotransmitter, the uptake of glutamate by astrocytes followed by its amination to glutamine, which is returned to the neurons and deaminated, implies a flux of ammonium (Benjamin and Quastel, 1975; Hassel et al., 1997). In bee retina, the main metabolic substrate of the neurons (photoreceptors) is alanine formed by amination of pyruvate in the predominant glial cells (“outer pigment cells”). The alanine is transferred to the photoreceptors and deaminated to pyruvate and the tissue releases ammonium (Tsacopoulos et al., 1994, 1997b; Coles et al., 1996). Uptake of ammonium into cells can be monitored continuously, but indirectly, by measuring the changes in intracellular pH (pH i ) that it causes. Ammonium has a pK a of z 9.2 in water (Sillén, 1964) so that at physiological pH (in the range 6.5–7.5) a fraction in the order of 1% is in the neutral NH 3 form. Nearly all cell membranes are permeable to NH 3 (but see Singh et al., 1995), so, when ammonium is applied outside a cell, NH 3 diffuses into it, combines with H 1 , and tends to raise pH i (Jacobs, 1940). In contrast, in astrocytes cultured from neonatal mouse, application of ammonium lowers pH i because there is an influx of NH 4 1 whose effect on pH i outweighs the effects of NH 3 fluxes (Nagaraja and Brookes, 1998). The glial cells in slices of bee retina also take up NH 4 1 (Coles et al., 1996), an observation that has been confirmed and extended on bundles of glial cells freshly dissociated from adult retinas (Marcaggi et al., 1999). Application of ammonium causes a fall in pH i that requires the presence of external Cl 2 and is blocked by loop diuretics such as bumetanide (Marcaggi et al., 1999). These observations suggest that NH 4 1 enters the glial cells by cotransport with Cl 2 on a transporter with functional similarities to the cation–chloride cotransporters present on many types of cells. The transport on the bee glial cells is not blocked in the absence of Na 1 (Marcaggi et al., 1999), indicating that the transport is of the K 1 -Cl 2 class rather than the Na 1 -K 1 -2Cl 2 class (see Race et al., 1999). Several cases have been described of cation-chloride Dr. Marcaggi’s present address is Department of Physiology, University College London, London WC1E 6BT, UK. Address correspondence to Dr. Païkan Marcaggi, Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK. Fax: 0044 171 413 8395; E-mail: [email protected] ✪ The online version of this article contains supplemental material. on July 6, 2017 jgp.rress.org D ow nladed fom http://doi.org/ 0.1085/jgp.116.2.125 Supplemental material can be found at: 126 Selectivity for NH 4 1 of a Cl 2 Cotransporter cotransporters, particularly in kidney, being able to transport NH 4 1 in the place of K 1 , although with a lower affinity (Kinne et al., 1986). However, in plant roots, transporters are known that are selective for NH 4 1 over K 1 (e.g., Kaiser et al., 1998) so such selectivity is a demonstrated biological possibility. We have found that uptake of NH 4 1 by the transporter in bee retinal cells is only moderately affected by external [K 1 ]. This suggested that the transporter might be the first to be described in an animal cell that is selective for NH 4 1 over K 1 and prompted us to make a quantitative estimate of its selectivity. Influx of NH 4 1 into a cell is generally associated with transmembrane fluxes of NH 3 (Boron and De Weer, 1976; see Fig. 2 C), so the relation between changes in pH i ( D pH i ) and NH 4 1 flux (F NH4 ) 1 is complex. We tackled the question of the NH 4 1 /K 1 selectivity in two stages. First, we deduced F NH4 from D pH i for relatively brief applications of ammonium. This required accurate absolute measurements of pH i and measurement of several other parameters: membrane permeability to NH 3 , intracellular buffering power, and the kinetics of pH i regulation. Use of this “cell model” showed a functional selectivity for NH 4 1 over K 1 . We then recorded pH i responses to longer and more complex NH 4 1 application protocols. By simulating these responses with a standard minimal model for a cotransport process, to which we added competitive inhibition, we estimated the NH 4 1 and K 1 affinities of the transporter molecule. M A T E R I A L S A N D M E T H O D S Intracellular pH (pH i ) in bundles of glial cells dissociated from the retina of the drone (male) Apis mellifera was measured by techniques developed from those described in Marcaggi et al. (1999). One record is shown (see Fig. 9 D) from an intracellular microelectrode recording of glial membrane potential in a slice of retina prepared and superfused with oxygenated Cardinaud solution, as described previously (e.g., Coles et al., 1996). Unless otherwise stated, results are given as mean 6 SD and the twotailed paired t test was used to determine P values. Errors of quotients were estimated by the calculus of errors (Abramowitz and Stegun, 1965). Dissociation Procedure and Loading of the Cells Bees were obtained from A. Dittlo (Villandraut) or J. Kefuss (Toulouse, France) and maintained on sugar water. A slice of drone head z 500m m thick was cut with a razor blade. The slice was incubated for 40 min in a 1.5 ml Eppendorf tube containing 1 ml oxygenated Cardinaud solution (see below) to which had been added 2 mg trypsin (T4665; Sigma-Aldrich). The slice was washed in Cardinaud solution lacking Ca 2 1 and Mg 2 1 and the retinal tissue dissected out and triturated. 150 m l of cell suspension was placed in the perfusion chamber (see below) whose floor consisted of a microscope cover slip coated with poly-llysine. The cells were allowed to settle for 10 min and then exposed to the acetoxymethyl ester of 29,79-bis(2-carboxyethyl)5(6)-carboxyfluorescein (BCECF-AM) (Molecular Probes, Inc.) at a concentration of 10 mM for 40 min. Measurement of Fluorescence The chamber was placed on the stage of an inverted microscope (Diaphot; Nikon) equipped with a 403 objective, photomultiplier detection, and dual wavelength excitation at 440 and 495 nm switched by liquid crystal shutters, as described in Coles et al. (1999). The stimulating light intensity was attenuated so that fluorescence from a bundle of loaded glial cells excited at 440 nm gave a signal of z10,000 photon counts s21, which remained stable for several hours. Dark noise plus autofluorescence from a bundle of unloaded cells was ,2,500 photon counts s21 for both excitation wavelengths and it was checked that this fluorescence was not affected by ammonium superfusion (n 5 4). This background fluorescence was automatically taken into account in the in situ pH calibration of each cell bundle (see below). The excitation pattern was usually: 440 nm, 100 ms; off, 20 ms; 495 nm, 600 ms; off 20 ms. To minimize the noise of the ratio, the signal resulting from excitation near the isosbestic point (440 nm) was averaged over several minutes before the PC computer calculated the ratio using a program available from Jean-Louis Lavie (University Bordeaux, Bordeaux, France).