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Perturbation of astroglial Slc38 glutamine transporters by NH4 + contributes to neurophysiologic manifestations in acute liver failure.
Hamdani EH
,
Popek M
,
Frontczak-Baniewicz M
,
Utheim TP
,
Albrecht J
,
Zielińska M
,
Chaudhry FA
.
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Ammonia is considered the main pathogenic toxin in hepatic encephalopathy (HE). However, the molecular mechanisms involved have been disputed. As altered glutamatergic and GABAergic neurotransmission has been reported in HE, we investigated whether four members of the solute carrier 38 (Slc38) family of amino acid transporters-involved in the replenishment of glutamate and GABA-contribute to ammonia neurotoxicity in HE. We show that ammonium ion exerts multiple actions on the Slc38 transporters: It competes with glutamine for the binding to the system N transporters Slc38a3 and Slc38a5, consequently inhibiting bidirectional astroglial glutamine transport. It also competes with H+ , Na+ , and K+ for uncoupled permeation through the same transporters, which may perturb astroglial intracellular pH, membrane potential, and K+ -buffering. Knockdown of Slc38a3 in mice results in cerebral cortical edema and disrupted neurotransmitter synthesis mimicking events contributing to HE development. Finally, in a mouse model of acute liver failure (ALF), we demonstrate the downregulation of Slc38a3 protein, impeded astroglial glutamine release, and cytotoxic edema. Altogether, we demonstrate contribution of Slc38 transporters to the ammonia-induced impairment of glutamine recycling between astrocytes and neurons, a phenomenon underlying acute ammonia neurotoxicity in the setting of ALF.
Figure 1. NH4 + inhibits selectively the substrate-induced currents associated with the system N transporters Slc38a3 and Slc38a5 and it penetrates through them uncoupled to transport. Xenopus (X.) laevis oocytes injected with in vitro transcribed mRNA for one of four Slc38 transporters were incubated with glutamine (5 mM) and subjected to two-electrode voltage-clamp electrophysiology. Voltage jumps were generated from a holding potential of −40 mV in increments of 20 mV ranging from −140 mV to +40 mV and voltage-current relationships were measured. A-D,Glutamine generates currents in oocytes expressing either of Slc38a1, Slc38a2, Slc38a3, or Slc38a5 transporters. Co-incubation with ammonium (1, 2, 4, or 8 mM) has only minor effects on the currents associated with Slc38a1 or Slc38a2 transporters. By contrast, ammonium abolishes glutamine-induced currents in oocytes expressing Slc38a3 or Slc38a5 transporters in a concentration-dependent manner. E, X. leavis oocytes injected for Slc38a3 and stimulated with glutamine show large inward currents at negative membrane potentials. Co-incubation with trimethylamine (TMA) has no impact on the glutamine-induced currents. F, Voltage-current measurements in X. leavis oocytes expressing Slc38a3 were performed in the absence of Na+ and glutamine. Rising ammonium concentrations increases the current magnitude and shifts the reversal potential toward positive values
Figure 2. Isoform-specific impact of ammonium on Slc38 transporter activity. A-B, Mammalian cells mock transfected or transfected for one of the Slc38 transporters were treated with or without ammonium and incubated with a radiolabeled substrate (2 mM MeAIB or 2 mM glutamine) followed by cell lysis in SDS and measurement of the accumulated substrate in a scintillation counter. A, MeAIB is accumulated to a significantly higher degree in Slc38a1 or Slc38a2 transfected cells compared with mock-transfected cells. Co-incubation with ammonium has no impact on transport activity. B, PS120 cells stably transfected for Slc38a3 or Slc38a5 accumulate significantly more glutamine than the mock-transfected control cells. Co-incubation with ammonium significantly reduces glutamine uptake in cells expressing Slc38a3 or Slc38a5. C, Slc38a3, Slc38a5, or mock transfected PS120 cells were loaded with radiolabeled glutamine or MeAIB for 1.5 hours followed by incubation in amino acid-free buffer with or without 5 mM NH4Cl. Net efflux was calculated as the concentration in the supernatant after 30 minutes over 0 minutes. MeAIB was used as a non-system N substrate control. Slc38a3 and Slc38a5 transfected cells release significantly more glutamine than mock-transfected cells. Co-incubation of the cells with ammonium significantly reduces glutamine efflux by both transporters. Note that MeAIB release is not increased by Slc38a3 or Slc38a5 transfected cells nor changed by pre-incubation in ammonium. In A-C all graphs show mean values with SEM. Significance were assessed by two-tailed t-tests and significant P-values are indicated by asterisks where *P < .05 and ***P < .001
Figure 3. Ammonium abolishes Slc38a3- and Slc38a5-mediated cellular alkalinization. Cultured Müller glia cells or PS120 cells stably transfected for Slc38a3 or Slc38a5 were loaded with the H+-sensitive dye BCECF and stimulated with glutamine in the absence or presence of ammonium prior to ratiometric analyses. A, Slc38a3 and Slc38a5 expressing rat Müller glia cells show increase in intracellular pH when stimulated with glutamine. B, pH trace from one representative Müller glia cell is shown. Glutamine induces alkalinization. Addition of NH4 + reduces pHi. The glutamine-induced alkalization is obliterated upon addition of glutamine in the presence of NH4 +. C-D, Slc38a3 and Slc38a5 expressing PS120 cells show glutamine-induced increase in intracellular pH which returns to baseline upon removal of glutamine. Upon co-application of glutamine with ammonium, the intracellular alkalinization is significantly reduced at 0.5 mM NH4 + and disappears at 1 mM NH4 + (effects of mere NH4 + are deducted). Representative traces are shown from one cell each
