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Recovery of hepatocytes from attack by the pore former amphotericin B
Abstract:
Sublytic amounts of the pore former Amphotericin B (AmB) induced transient movements of Na and K ions across the hepatocyte plasma membranes without altering the intracellular free Ca ion concentration. The presence of 1-5 microM-AmB induced leakage of up to 80% of the intracellular K+ within 3 min, followed by Na+ entry without loss of cell viability. A repair process occurred after 3-10 min, which restored the initial cationic concentrations. Progressive binding of AmB to the cells could be observed by following the disappearance of the intense excitonic dichroic doublet of free AmB. It was shown that the amount of AmB binding, responsible for the Na+ and K+ movements, was low (approx. 16% of total AmB). The recovery process occurred when higher amounts of AmB bound to the cells, and was mediated by Na+/K+-ATPase. The c.d. spectrum of AmB bound to isolated hepatocyte plasma membranes, indicated that during this step AmB formed a complex with cholesterol, similar to that formed by the binary mixture in water.
Insights
Sublytic Amphotericin B (AmB) causes transient ion shifts in liver cells without affecting calcium levels. A repair mechanism, mediated by Na+/K+-ATPase, restores ion balance and cell viability.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Amphotericin B (AmB) is a known antifungal agent that interacts with cell membranes.
- Its precise effects on ion transport and cell viability at sublytic concentrations require further elucidation.
Purpose of the Study:
- To investigate the impact of sublytic Amphotericin B concentrations on ion flux in hepatocytes.
- To characterize the cellular response and recovery mechanisms following AmB-induced membrane perturbation.
Main Methods:
- Hepatocytes were treated with varying concentrations of Amphotericin B.
- Ion concentrations (Na+, K+, Ca2+) were monitored using spectroscopic techniques.
- AmB binding to cell membranes was assessed via circular dichroism (c.d.) spectroscopy.
- The role of Na+/K+-ATPase in the recovery process was investigated.
Main Results:
- Sublytic AmB induced rapid and transient leakage of intracellular potassium (K+) and subsequent sodium (Na+) influx.
- These ion movements occurred without altering intracellular free calcium (Ca2+) levels.
- A significant portion of AmB binding (approx. 16%) was responsible for the initial ion flux.
- Cell viability was maintained, and a repair process restoring cationic concentrations was observed within 3-10 minutes.
- The recovery was mediated by the Na+/K+-ATPase and associated with higher AmB binding.
- AmB formed a complex with cholesterol in the hepatocyte plasma membrane.
Conclusions:
- Sublytic Amphotericin B causes transient ion permeability in hepatocytes.
- Hepatocytes possess a robust repair mechanism involving Na+/K+-ATPase to restore ion homeostasis and viability.
- AmB-cholesterol complex formation is implicated in the membrane interactions during the recovery phase.