Table 4.
Fluorescent probe studies of plasma membrane permeabilization in relation to glycine cytoprotection
| Cell type | Injury model | Pore probes | Behavior without glycine | Behavior with glycine |
|---|---|---|---|---|
| MDCK and LLC-PK1 (renal epithelial) [40] and human umbilical vein endothelial [41] | Uncoupler [carbonyl cyanide m-chlorophenylhydrazone (CCCP)] + ionomycin |
Propidium (416 Da) Fluorescein (332 Da) |
Nearly all cells develop LDH release, fluorescein release, and propidium uptake | LDH release, fluorescein release, and propidium uptake all prevented |
| MDCK [99] | Uncoupler (CCCP) + ionomycin in 100 nM Ca2+ medium |
Propidium (416 Da) Fluorescent dextrans—4 kDa (2–3 nm molecular diameter), 70 kDa (10–15 nm), 2000 kDa (54 nm) |
Majority of cells leak LDH and become progressively positive for propidium over 120 min and similarly permeable to 4 kDa dextran. Heterogeneous loss of permeability to 70 kDa dextrans with many propidium-positive cells that completely exclude 70 kDa dextran. All cells exclude 2000 kDa dextran | LDH leak blocked. Rare propidium-positive cells. All dextrans including 4 kDa excluded |
| MDCK [145] | Uncoupler (CCCP) + ionomycin in 100 nM Ca2+ medium |
Calcein (623 Da) Ethidium homodimer (717) |
Majority of cells had leaked LDH and calcein and taken up ethidium homodimer at 2 h | LDH and calcein leaks and ethidium homodimer uptake blocked |
| LLC-PK1 [85] | H2O2 | Trypan blue (961 Da) | Cells display LDH release and Trypan blue uptake at 2 h | LDH release suppressed, but no change in failure of Trypan blue uptake |
| Hepatic sinusoidal endothelial in primary culture [43] | Chemical hypoxia (cyanide) |
Propidium (416 Da) Calcein (623 Da) Lucifer Yellow (443 Da) Fluorescent dextrans (40–2000 kDa) |
Cells become permeable to calcein and start forming blebs at 135–180 min, followed after 30–60 min by uptake of propidium and 40 kDa dextran and, to a lesser extent, 2000 kDa dextran. Uptake of propidium and all sizes of dextrans is simultaneous | Slows but does not prevent the calcein and Lucifer yellow uptake. Prevents uptake of propidium and 40 and 2000 kDa dextran |
| Freshly isolated kidney proximal tubules [167] | Hypoxia |
Propidium (416 Da) Phallacidin (846 Da) Fluorescent dextrans (3 and 70 kDa) Trypan blue (961 Da) |
Rapid (5–15′) uptake of 20 μM propidium followed after 15′ by LDH release. Phallacidin uptake delayed relative to propidium At 30 min, when LDH release was 50 %, all cells were permeable to 3 kDa dextran, but only a few were permeable to 70 kDa dextran |
No effect on propidium uptake, but all cells remain impermeable to 3 and 70 kDa dextran |
| Bovine aortic endothelial cells [44, 169] | Maitotoxin |
Ethidium (359 Da) Propidium (416 Da) Transfected GFP concatemers (27–162 kDa) |
Initial Ca2+ increase-dependent induction of slow ethidium uptake followed by accelerated ethidium uptake coinciding with LDH release, propidium uptake, and release of GFP concatemers without detectable size dependence in latency to release or rate of release | No effect on Ca2+ increase or initial slow phase of ethidium uptake. Prevents the rapid phase of ethidium uptake, propidium uptake, and release of LDH and GFP concatemers |
| Bovine aortic endothelial cells [168] | Palytoxin |
Ethidium (359 Da) Yo-Pro-1 (377 Da) Propidium (416 Da) Transfected GFP (27 kDa) |
Initial ouabain-sensitive Ca2+ increase and slow ethidium uptake, but no uptake of Yo-Pro-1 or propidium. After variable delay, rapid ethidium uptake coincides with Yo-Pro-1 and propidium uptake and GFP loss | No effect on initial slow ethidium uptake, but blocks the subsequent rapid phase of ethidium uptake and GFP loss |
| BAC1.2F5 macrophages [92] | Maitotoxin | Yo-Pro-1 (377 Da) | Early Ca2+-dependent induction of slow Yo-Pro uptake followed by accelerated Yo-Pro uptake coinciding with LDH release | No effect on initial slow phase of Yo-Pro uptake. Prevents the rapid phase of Yo-Pro and release of LDH |
|
J774A.1 macrophages [52] Bone marrow-derived macrophages, dendritic cells [91] |
S. typhimurium and anthrax lethal toxin-induced pyroptosis |
Ethidium (359 Da) Ethidium homodimer-2 (793 Da) |
2.5 h of infection induces caspase-1-dependent permeability to ethidium, but not to ethidium homodimer-2; cell swelling and LDH release follow | Prevents swelling and LDH release, but not the initial ethidium uptake. PEG 1450 (2.4 nm molecular diameter) and PEG 2000 (2.8 nm), but not PEG 200 (1.12 nm), also limit LDH release |