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- Materials and methods
Inorganic phosphate (Pi) is an important polyanion needed for ATP synthesis and bone formation. As it is found at millimolar levels in plasma, it is usually incorporated as a constituent of artificial CSF formulations for maintaining brain slices. In this paper, we show that Pi limits the extracellular zinc concentration by inducing metal precipitation. We present data suggesting that amino acids like histidine may counteract the Pi-induced zinc precipitation by the formation of soluble zinc complexes. We propose that the interplay between Pi and amino acids in the extracellular space may influence the availability of metals for cellular uptake.
An essential step in the process of unraveling the mechanisms of action potential generation, synaptic transmission, and muscular contraction was identifying the nature of the ionic species necessary for the operation of excitable cells (Burton 1975). This began with Ringer’s demonstration (Ringer 1883) that extracellular calcium was necessary for cardiac contraction and set in motion the process of formulating media used to sustain cells in vitro. An important step was Krebs and Henseleit’s (1932) realization that the constituents of normal plasma would be a good starting point for formulating physiological salines. Building on these foundations, McIlwain developed the in vivo brain slice preparation, one that has been enormously influential in the progress of neuroscience (Li and McIlwain 1957).
Inorganic phosphate (Pi, orthophosphate) is an essential ion in living organisms playing indispensable roles in ATP synthesis and bone mineralization, among other processes. Pi exists in two predominant forms at physiological pH; HPO42−and H2PO4− at ∼4 : 1 ratio. Intracellular Pi is sustained at a concentration of about 2 mM in most mammals and is a key determinant of the free energy available from ATP hydrolysis (Erecinska and Silver 1989). Plasma Pi levels vary considerably in different vertebrates (Furman et al. 1997). In human plasma, the normal Pi level is ∼1.1 mM but fluctuates more widely than calcium, and exhibits circadian variations. The concentration of Pi in the CSF of mammals is ∼0.4 mM; however, little is known about the concentration in the interstitial space.
Little information is available on the mechanism of Pi uptake into cells of the CNS. A protein initially identified as a Pi transporter (Ni et al. 1994) was subsequently shown to carry glutamate into synaptic vesicles (Bellocchio et al. 2000; Takamori et al. 2000) and its Pi transporting capabilities are uncertain. Pi transport has been well characterized in the kidneys where it is transported by members of the Slc34 (Murer et al. 2004) and Slc20 (Collins et al. 2004) families for monovalent and divalent species, respectively, in sodium-dependent processes.
There are abundant opportunities for solid minerals to form from the complex mixtures of ions in and around cells, particularly between metals and polyanions like Pi. In a solution with known concentrations of ions it is possible to predict the formation of precipitates from the solubility products (Ksp) for the various ion combinations. As a thermodynamic parameter, the Ksps however give no indication of how fast the precipitate takes to form. One common form hydroxyapatite (Ca10(PO4)6(OH)2) takes a long time, others like hopeite (Zn3(PO4)2·4H2O) form within a few milliseconds.
Zinc is found at a high concentration in certain glutamatergic vesicles within the mammalian forebrain and it has been proposed to be released and act as a neuromodulator (Smart et al. 2004; Paoletti et al. 2009). There is a potential chemical impediment to the free release of zinc ions, namely, that zinc–phosphate has a very low solubility product (9.1 × 10−36 M5), which limits the concentration of free zinc ions in a solution with a high concentration of Pi. In this communication, we show that in brain slices the extracellular free zinc concentration is indeed limited by precipitation. Moreover, we demonstrate that this limitation can be overcome by the provision of amino acids like histidine that increases the solubility of the metal.
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- Materials and methods
We have shown here that in brain slices, the presence of phosphate in physiological saline limits the concentration that soluble zinc can reach after metal application by the formation of zinc phosphate precipitates. In addition, we have shown that amino acids like histidine can increase zinc solubility in the presence of Pi, but they do not play a direct role in the transport of zinc. These results have clear implications for experiments where exogenous zinc is applied to brain slices. For example, it accounts for Molnar and Nadler’s (2001) observation that the presence of phosphate inhibited the action of exogenous zinc on GABA receptors. Our results also have perhaps less obvious implications for zinc release and uptake in vivo that we will discuss below. Moreover, our experiments have cast a spotlight on a rather underappreciated yet ubiquitious anion, Pi.
It is widely believed that synaptic zinc acts as a neuromodulator, being released during exocytosis and then diffusing into the synaptic cleft (Frederickson et al. 2005). In contrast, our laboratory has provided evidence which conflicts with this idea and instead we have suggested that rather than being released, zinc is presented to the extracellular space while bound to exocytosed vesicular proteins (Kay 2003; Kay and Tóth 2006). We have termed this scenario ‘externalization’ in contrast to simple release (Kay and Toth 2008). The presence of extracellular Pi poses a problem for the zinc release hypothesis, as precipitates could form if large amounts of zinc are released. Whether they do form will depend on the concentration of zinc chelators, small ligands, macromolecules, Pi, zinc, and pH. We cannot, however, exclude the existence of zinc chelators more powerful than those so far identified.
The concentration of glutamate within vesicles is estimated to be ∼300 mM, but after exocytosis it declines very rapidly as the molecule diffuses within the synaptic cleft (Barbour and Hausser 1997). Glutamate has a low affinity for zinc; nevertheless at high concentrations, it can solubilize zinc. For example, at a zinc concentration of 100 μM in normal saline if the glutamate concentration is above ∼30 mM no precipitate forms, however below this concentration progressive amounts of precipitate form with half the zinc being precipitated with ∼14 mM glutamate (minteqa2 calculations).
Histidine has been proposed to augment the transport of zinc by forming a 2 : 1 complex (Sivarama Sastry et al. 1960) that is either transported as a unit or hands zinc off to a transporter. Histidine has been found to facilitate zinc transport in erythrocytes (Aiken et al. 1992) and in the intestines of rats (Wapnir et al. 1983), trout (Glover and Hogstrand 2002), and lobsters (Conrad and Ahearn 2007). However, our results suggest that in brain slices histidine simply increases the solubility of zinc and does not serve to facilitate the transport of zinc.
Our finding that histidine does not augment zinc transport in PFS suggests that under these conditions there is little phosphate in the extracellular space of brain slices. Similarly, because histidine increases zinc transport in normal saline, this indicates that the amino acid concentration is rather low in slices. In human CSF, the total amino acid concentration is ∼700 μM (∼500 μM glutamine) and the histidine concentration ∼12 μM (Davson et al. 1993; Wishart et al. 2008). This concentration of histidine is too low to increase the solubility of zinc in the presence of Pi.
However, the fact that in normal saline histidine augments zinc transport does not imply that there are no amino acids in the extracellular space. It could be that the zinc–phosphate particles do not penetrate through the extracellular space, and too little amino acid is likely to leach out to solubilize it. It is likely that the amino acid levels in the extracellular space of brain slices are diminished as they diffuse into the bathing solution. However, there is evidence that extracellular glutamate and perhaps glycine levels remain elevated in slices (Sah et al. 1989).
It does not seem to have been widely appreciated that Pi is an essential component of the extracellular medium that goes beyond its role in pH regulation. If Pi is removed from saline bathing neocortical cells, the intracellular ATP and Pi levels remain stable for 30 min but both decline to ∼60% of control levels after an hour (Glinn et al. 1997). Furthermore, synaptosomes derived from chronically phosphate-deprived rats show an increase in cytosolic calcium and a decrease in ATP (Massry et al. 1991).
Controlled precipitation plays an important role in skeleton formation and other biomineralization processes (Dorozhkin and Epple 2002). On the other hand, the uncontrolled formation of insoluble aggregates plays a prominent role in the pathogenesis of atherosclerosis (Giachelli et al. 2001) and may do so in a number of neuropathologies, including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. We would like to suggest that the formation of inorganic precipitates could serve as a nucleus for the accretion of molecules and ions. However, if one is to implicate precipitation in a neuropathology it is important to exclude aggregates that arise during the analysis. In this regard, it is instructive to consider the case of entities termed ‘nanobacteria’ that on closer examination turned out to be calcium carbonate particles (Martel and Young 2008).
Aggregates of material with a predominantly inorganic basis have been described in a number of neuropathologies that have for the most part been ascribed to the precipitation of calcium. The development of calcifications within the brain has been noted in the case of ischemia and exitotoxicity (Mahy et al. 1999); no zinc was found in this case (Mahy N., personal communication). Calcifications have also been described in the case of Fahr’s syndrome in the basal ganglia (Bouras et al. 1996), spasmodic dysphonia (Simonyan et al. 2008), and in Urbach–Wiethe disease in the amygdala (Thornton et al. 2008); some zinc is found in the case of Fahr’s syndrome. It is also worth noting that excess intracellular Pi may lead to the precipitation of calcium as it does in the case of skeletal muscle sarcoplasmic reticulum, limiting calcium mobilization (Dutka et al. 2005).
Although Pi is usually incorporated in solutions used for sustaining brain slices, there are cases where its omission is seemingly without effect (Miles 1990), but this has not been studied systematically. In cultured neurons, many investigators leave out phosphate from the minimal solutions used to perform physiological experiments but preserve it in the media used to culture the cells.
The saline formulations used currently for sustaining brain slices are based on the concentrations of ions in plasma. The plasma concentration of Pi is around 1.1 mM in humans and tends to be higher in other animals. In rats, the plasma Pi concentration is 3.2 ± 0.1 mM and in CSF 0.47 ± 0.01 mM (Mulroney et al. 2004). The latter is close to the concentration in humans and it is likely but not certain that the extracellular concentration is similar. Most mammalian slice ACSF formulations have Pi at a concentration of around 1 mM; to mimic CSF more closely it may be worthwhile shifting to a concentration of ∼0.5 mM.
It is worth considering whether Pi might play roles other than that of the substrate for ATP synthesis. For example might it act as an allosteric regulator of ion channels and transporters? Moreover, the outward directed Pi gradient could be employed in carrier-mediated mechanisms to transport ions, although none have thus far been identified.
There is a pressing need for experiments to determine the range of concentrations of Pi in the extracellular space and to determine the effect of changes in Pi on neuronal and synaptic activity. NMR measurements in the intact brain can distinguish between extra and intracellular Pi and may provide a means for assessing the extracellular Pi concentration (Gilboe et al. 1998).
To the best of our knowledge, there have been no systematic studies of the effect of phosphate-free ACSF on synaptic transmission. We found that phosphate removal for up to 5.5 h had no effect on transmission at the Schaffer collateral CA1 synapse. This suggests that intact neuronal tissue either has considerable reserves of Pi or that it is endowed with an exceptional capacity to reclaim Pi that passes into the extracellular space. Until it becomes feasible to measure extracellular Pi, it is not possible to say whether or not Pi levels are sustained in slices held in PFS.
It is clear that the role of Pi extends beyond that of a pH buffering agent. There is little information on the mechanisms to control the intra and extracellular Pi levels, which could have a profound impact on the precipitation of metals. Moreover, the levels of amino acids, like histidine could play an important role in formation of metal–phosphate precipitates.
Perhaps because phosphate has been identified as part of the pH buffer system of physiological salines, it has not received much attention as a necessary component of media. Pi seems to have become part of the scenery when in fact it is a significant supporting player in cellular biology.