Ammonium chloride (NH4Cl)is as a well-known lysosomotropic autophagy inhibitor that alkalizes acidic pH.NH4Cl acts by disturbing the endo/lysosomal and autophagy/lysosomalpathway through neutralizing the low pH. This process inhibits degradation stepby blocking lysosomes.
To measure autophagic flux, LC3 turnover assays areestablished, which are based on the observation that LC3 is degraded inautolysosomes. When cells are treated with autophagy inhibitors such as NH4Cl,the degradation of LC3-II is blocked causing it accumulate. The differences inthe quantity of LC3-II between samples in the presence and absence of lysosomalinhibitors represent the amount of LC3 which is delivered to lysosomes fordegradation, this what was described earlier as autophagic flux. Any autophagy inhibitor willusually result in an increase in LC3-II puncta (Mizushima et al.
, 2010). To make sure thatthe autophagosomes are not degraded before true LC3-II measurements areestablished, this alkalization factor inhibits the autophagic pathway beforethe degradation step. Given that autophagy is part of the cellular stressresponse, it is not unexpected that this process can also influence decisionmaking on cell death (Martin,2011, Muñoz-Pinedo andMartin, 2014). Infact, excessive autophagy leads to increased autophagosome synthesis and may beassociated with decreased lysosomal activity thus remain unfused, and theiraccumulation is useless for autophagy. I one study it was found thataccumulation of autophagosomes compromised cell viability whereas experimentswith depleted autophagosomes did not (Robert W.) This can be compared to thep53-dependent cell response, which is primarily concerned with coordinating DNAdamage-associated repair also resulting in apoptosis above a certain thresholdof p53 activation.
This makes decent biological sense, as cells with extremeDNA damage are unsafe to repair due to the potential for the development ofcancer. (Muñoz-Pinedo,) Autosis is one of three types of cell death which are grouped based onmorphological criteria. Type I programmed cell death (apoptosis), type IIprogrammed cell death (Autosis or Autophagic-cell death), type III unprogrammedcell death (necrosis), the latter being an uncontrolled and non-physiologicaltype of cellular death. Autosisis a non-apoptotic novel form of autophagy gene-dependent cell death, which ismediated by the Na+, K+, ATPase pump. The morphological characteristics of thiscell death are predominantly enhanced cell-substrate adhesion, focal ballooningof the perinuclear space, and dilation and disintegration of the endoplasmicreticulum (ER).
(Liu, Y, and B Levine. ) This cell death occurs under certainexperimental conditions, triggered by treatment with autophagy-inducingpeptides, starvation and hypoxia and in vivo types of ischemia. Thereare contradictory reports that autophagy can be both oncogenic and tumorsuppressive. This presents a potential difficulty for using inhibitors ofautophagy to treat cancer among other diseases, as the effect of inhibitingautophagy in different situations is hard to predict. (Macintosh, Robin L).