Aquaporin-8-facilitated mitochondrial ammonia transport

Lipopolysaccharide impairs hepatocyte ureagenesis from ammonia: Involvement of mitochondrial aquaporin‐8

“…Control and LPS hepatocytes were incubated with 1 mM 15 N-labeled ammonium chloride (99% enriched in 15 N, Cambridge Isotope Laboratories, Andover, MA) at 37°C for 4 h in the absence or presence of 1 lM glucagon. The culture medium was then aspirated, centrifuged at 500g for 5 min at 4°C, and lyophilized.…”

“…The culture medium was then aspirated, centrifuged at 500g for 5 min at 4°C, and lyophilized. The lyophilizate was resuspended in 0.5 ml of dimethyl sulfoxide-d 6 (99.9 atom% D) (Cambridge Isotope Laboratories) containing 0.02 ml of formamide [natural abundance (0.365%) in 15 N] (Promega), that was used as internal standard. 15 N-labeled urea synthesis was determined by nuclear magnetic resonance spectroscopy, as previously described [17].…”

“…For measurement of formamide permeability, mitochondria were subjected to a 150 mM inwardly directed gradient of formamide as previously reported [15]. The time course of mitochondrial volume change was followed from changes in intensity of scattered light intensity at 20°C at the wavelength of 450 nm by using a SX.18MVR stopped-flow spectrometer (Applied Photophysics, Surrey, UK), which has a 1.3-ms dead time and 99% mixing efficiency in <1 ms.…”

“…The K i of swelling was used as an indirect biophysical parameter for assessment of formamide mitochondrial permeability. The corresponding solute permeability coefficients were not calculated because of biophysical (i.e., presence of two membranes in series) and morphological (i.e., rod-shaped structures of heterogeneous size) constraints of mitochondria [15].…”

“…The expression of mitochondrial AQP8 (mtAQP8) seems to be greater in the periportal area of the hepatic lobule [13,14]. There is experimental evidence suggesting that mtAQP8 facilitates the ammonia transport across the IMM [15][16][17]. We recently reported that hepatocyte mtAQP8 is involved in the mitochondrial uptake of ammonia and its metabolism into urea [17].…”