Mechanisms of Pancreatic Injury Induced by Basic Amino Acids Differ Between L-Arginine, L-Ornithine, and L-Histidine

Abstract

Pancreatic acinar cells require high rates of amino acid uptake for digestive enzyme synthesis, but excessive concentrations can trigger acute pancreatitis (AP) by mechanisms that are not well understood. We have used three basic natural amino acids L-arginine, L-ornithine, and L-histidine to determine mechanisms of amino acid-induced pancreatic injury and whether these are common to all three amino acids. Caffeine markedly inhibited necrotic cell death pathway activation in isolated pancreatic acinar cells induced by L-arginine, but not L-ornithine, whereas caffeine accelerated L-histidine-induced cell death. Both necroptosis inhibitors of RIPK1 and RIPK3 and a necroptosis activator/apoptosis inhibitor z-VAD increased cell death caused by L-histidine, but not L-arginine or L-ornithine. Cyclophilin D knock-out (Ppif^-/-) significantly attenuated cell death induced by L-histidine, but not L-arginine, or L-ornithine. Allosteric modulators of calcium-sensing receptor (CaSR) and G-protein coupled receptor class C group 6 member A (GPRC6A) had inhibitory effects on cell death induced by L-arginine but not L-ornithine or L-histidine. We developed a novel amino acid-induced AP murine model with high doses of L-histidine and confirmed AP severity was significantly reduced in Ppif^-/- vs. wild type mice. In L-arginine-induced AP neither Ppif^-/-, caffeine, or allosteric modulators of CaSR or GPRC6A reduced pancreatic damage, even though CaSR inhibition with NPS-2143 significantly reduced pancreatic and systemic injury in caerulein-induced AP. These findings demonstrate marked differences in the mechanisms of pancreatic injury induced by different basic amino acids and suggest the lack of effect of treatments on L-arginine-induced AP may be due to conversion to L-ornithine in the urea cycle.

Keywords: G-protein coupled receptors class C; acute pancreatitis; amino acids; caffeine; cyclophilin D; mitochondria; necroptosis.

FIGURE 1

Effect of caffeine on L-arginine-induced acute pancreatitis in vivo and on cell death caused by basic amino acids in vitro. Mice received intraperitoneal injections of L-arginine (pH 7.4, 2 × 4 g/kg, at 1 h interval) with or without concomitant caffeine administration (7 × 25 mg/kg, at hourly intervals) begun at 24 and 48 h after the first L-arginine injection and sacrificed at 72 h: (A) representative images of pancreatic histopathology changes (H&E, ×200, scale bar 50 μm) and scores – (B) overall, (C) oedema, (D) inflammation, and (E) necrosis. (F) Effect of caffeine (2 and 5 mM) on necrotic cell death pathway activation (presented as time to half-maximal response [HMR] of propidium iodide [PI] uptake) of freshly isolated mouse pancreatic acinar cells caused by basic amino acids (all at 20 mM): (i) L-arginine, (ii) L-ornithine, and (iii) L-histidine. ^∗p < 0.05 vs. L-arginine or L-histidine treatment only. Values are means ± SEM from ≥6 experiments/group (in vitro) or mice/group (in vivo).

FIGURE 2

Effect of cyclophilin D knock-out on cell death caused by basic amino acids in vitro and L-histidine-induced acute pancreatitis in vivo. (A) Effect of cyclophilin D knock-out (Ppif^-/-) in comparison with wild type (Wt) on necrotic cell death pathway activation (presented as time to half-maximal response [HMR] of propidium iodide [PI] uptake) of freshly isolated mouse pancreatic acinar cells caused by basic amino acids (all at 20 mM): (i) L-arginine, (ii) L-ornithine, and (iii) L-histidine (^∗p < 0.05 vs. L-histidine treatment only). Mice received intraperitoneal injections of 7% L-histidine (pH 7.4, 2 × 4 g/kg, at 1 h intervals) and were sacrificed at indicated time points after the first L-histidine injection: (B) representative images of pancreatic histopathology changes (H&E, ×200, scale bar 50 μm) and scores – (C) overall, (D) oedema, (E) inflammation, and (F) necrosis (^∗p < 0.05 vs. saline control, L-histidine 8 h or L-histidine 12 h, ^†p < 0.05 vs. L-histidine 24 h, ^#p < 0.05 vs. L-histidine 48 h). Values are means ± SEM from 3–6 experiments/group (in vitro) or mice/group (in vivo).

FIGURE 3

Comparison of the effect of cyclophilin D knock-out on acute pancreatitis caused by L-arginine and L-histidine. Cyclophilin D knock-out (Ppif^-/-) or wild type (Wt) mice received either intraperitoneal injections of either 8% L-arginine or 7% L-histidine (both: pH 7.4, 2 × 4 g/kg, at 1 h intervals) and were sacrificed at 72 h after the first L-arginine/L-histidine injection: (A) overall, (B) oedema, (C) inflammation and (D) necrosis histopathological scores; (E) serum amylase, (F) pancreatic trypsin activity, (G) pancreatic myeloperoxidase (MPO) activity and (H) lung MPO activity. ^∗p < 0.05 vs. L-histidine with Wt mice. Values are means ± SEM of 6–8 animals per group.

FIGURE 4

Effect of allosteric modulators of GPCR Class C on pancreatic acinar cell death caused by basic amino acids in vitro and on L-arginine-induced acute pancreatitis in vivo. (A) Images of immunohistochemistry staining against CaSR and GPRC6A in mouse and human fixed pancreatic acinar cells. (B) Effect of calcilytic NPS-2143 (1 μM), GPRC6A antagonist Cpd1 (50 μM), and calcimimetic calindol (1 μM) on necrotic cell death activation (presented as time to half-maximal response [HMR] of propidium iodide [PI] uptake) caused by basic amino acids (all at 20 mM): (i) L-arginine, (ii) L-ornithine, and (iii) L-histidine. Mice received intraperitoneal injections of either 8% L-arginine (pH 7.4, 2 × 4 g/kg, at 1 h interval) and were sacrificed at 72 h after the first L-arginine injection: (C) effects of calcilytic NPS-2143 and calcimimetic calindol in vivo, representative images of pancreatic histopathology changes (H&E, ×200, scale bar 50 μm) and scores – (D) overall, (E) oedema, (F) inflammation, and (G) necrosis. ^∗p < 0.05 vs. L-arginine or L-histidine treatment only. Values are means ± SEM from ≥6 experiments/group (in vitro) or mice/group (in vivo).

FIGURE 5

Effect of allosteric modulators of GPRC6A on L-arginine-induced acute pancreatitis. Mice received intraperitoneal injections of either 8% L-arginine (pH 7.4, 2 × 4 g/kg, at 1 h interval) and were sacrificed at 72 h after the first L-arginine injection: (A) Effects of GPRC6A antagonist Cpd1 in vivo, representative images of pancreatic histopathology (H&E, ×200, scale bar 50 μm) and scores – (B) overall, (C) oedema, (D) inflammation, and (E) necrosis. Values are means ± SEM from six mice/group.

FIGURE 6

Effect of NPS-2143 on caerulein-induced acute pancreatitis. Mice received intraperitoneal injections of caerulein (7 × 50 μg/kg, at 1 h interval) and were sacrificed at 12 h after the first caerulein injection: (A) representative pancreatic histopathology (H&E, ×200, scale bar 50 μm) and scores – (B) overall, (C) oedema, (D) inflammation, and (E) necrosis; (F) serum amylase, (G) pancreatic trypsin activity, (H) pancreatic myeloperoxidase (MPO) activity, and (I) serum interleukin (IL-6) levels. p < 0.05 vs. caerulein treatment only. Values are means ± SEM from six mice/group.