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Regulation of lysosomal fusion during deprivation-induced autophagy in perfused rat liver.

ABSTRACT: Previous studies have demonstrated that autophagic vacuoles induced in hepatocytes of the perfused rat liver can be effectively separated from secondary lysosomes in self-generating gradients of colloidal silica/polyvinylpyrrolidone. Here we show that, as autophagy is increased by graded amino acid deprivation, the lysosomal marker N-acetyl-beta-D-glucosaminidase shifts progressively from dense to buoyant gradient fractions, an effect attributable to enhanced lysosome-autophagosome fusion and the formation of additional autolysosomes. Both the amount of the shift and the aggregate volume of autolysosomes were directly proportional to the increase in protein degradation above basal. Because the total quantity of protein sequestered in the vacuoles is also a direct function of deprivation-induced proteolysis, the relative specific activity of the marker enzyme in autolysosomes (enrichment) would be expected to remain constant over the full range of deprivation; numerical estimates of enrichment, in fact, did not differ from the mean (43.9) by more than 5%. The close correspondence between cytoplasmic sequestration and fusion was maintained, despite a greater than 3-fold increase in the average volume of individual vacuoles. This suggests the existence of an intrinsic regulatory mechanism which limits the number of fusions per vacuole, on the basis of information derived from vacuolar volume.

SUBMITTER: Surmacz CA

PROVIDER: S-EPMC1147726 | biostudies-other | 1987 Mar

REPOSITORIES: biostudies-other

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Project description:Pharmacological preconditioning (PC) and postconditioning (PoC), for example, by treatment with the ?2-adrenoreceptor agonist Dexmedetomidine (Dex), protects hearts from ischemia-reperfusion (I/R) injury in experimental studies, however, translation into the clinical setting has been challenging. Acute hyperglycemia adversely affects the outcome of patients with myocardial infarction. Additionally, it also blocks cardioprotection by multiple pharmacological agents. Therefore, we investigated the possible influence of acute hyperglycemia on Dexmedetomidine-induced pre- and postconditioning. Experiments were performed on the hearts of male Wistar rats, which were randomized into 7 groups, placed in an isolated Langendorff system and perfused with Krebs-Henseleit buffer. All hearts underwent 33 min of global ischemia, followed by 60 min of reperfusion. Control (Con) hearts received Krebs-Henseleit buffer (Con KHB), glucose (Con HG) or mannitol (Con NG) as vehicle only. Hearts exposed to hyperglycemia (HG) received KHB, containing 11 mmol/L glucose (an elevated, but commonly used glucose concentration for Langendorff perfused hearts) resulting in a total concentration of 22 mmol/L glucose throughout the whole experiment. To ensure comparable osmolarity with HG conditions, normoglycemic (NG) hearts received mannitol in addition to KHB. Hearts were treated with 3 nM Dexmedetomidine (Dex) before (DexPC) or after ischemia (DexPoC), under hyperglycemic or normoglycemic conditions. Infarct size was determined by triphenyltetrazoliumchloride staining. Acute hyperglycemia had no impact on infarct size compared to the control group with KHB (Con HG: 56 ± 9% ns vs. Con KHB: 56 ± 7%). DexPC reduced infarct size despite elevated glucose levels (DexPC HG: 35 ± 3%, p < 0.05 vs. Con HG). However, treatment with Dex during reperfusion showed no infarct size reduction under hyperglycemic conditions (DexPoC HG: 57 ± 9%, ns vs. Con HG). In contrast, hearts treated with mannitol demonstrated a significant decrease in infarct size compared to the control group (Con NG: 37 ± 3%, p < 0.05 vs. Con KHB). The combination of Dex and mannitol presents exactly opposite results to hearts treated with hyperglycemia. While DexPC completely abrogates infarct reduction through mannitol treatment (DexPC NG: 55 ± 7%, p < 0.05 vs. Con NG), DexPoC had no impact on mannitol-induced infarct size reduction (DexPoC NG: 38 ± 4%, ns vs. Con NG). Acute hyperglycemia inhibits DexPoC, while it has no impact on DexPC. Treatment with mannitol induces cardioprotection. Application of Dex during reperfusion does not influence mannitol-induced infarct size reduction, however, administering Dex before ischemia interferes with mannitol-induced cardioprotection.

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Regulation of lysosomal fusion during deprivation-induced autophagy in perfused rat liver.

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