Assessment of membrane permeability in primary cultures of neurons and glia in response to osmotic perturbation

Abstract

In primary cultures of neurons and astrocytes from rat brain, the cellular contents of K+ and Na+ were used to assess plasma membrane permeability in response to osmotic shock. In contrast to trypan blue exclusion, the measure of ionic content reflected both transient and permanent membrane damage, and was also applicable to aggregates of cells. In steady state, the neurons and glia exhibited a K+ to Na+ ratio of 3-5, and the mean cellular contents (mu Eq/mg protein) of K+ and Na+ were 0.72 and 0.17 for astrocytes and 0.78 and 0.23 for neurons. Both hypo- and hypertonicity resulted in marked efflux of cell K+, but elevation of cellular Na+ occurred only under severe hypertonic conditions. Relative to neurons, astrocytes displayed considerably higher resistance to osmotic shock. During subsequent isotonic incubation, these cells were able to completely recover from transient membrane damage caused by a 10-min exposure to fourfold hypertonicity. Permanent changes in glial permeability were obtained only after a 20-min hypertonic shock. In contrast, 5 min hypertonic treatment of neurons decreased the ratio of cellular K+ to Na+ from 4.5 to 1. This ratio was restored twofold by isotonic incubation, but decreased permanently to below 1 after 10 min of hypertonic shock. The results describe marked differences in the osmotic fragility of neurons and glia and demonstrate that the determination of cellular K+ and Na+ provides a sensitive and accurate indicator of membrane permeability in neural cells propagated as surface-growing cultures. The approach has wide-ranging applicability.

The 43-kDa neuronal growth-associated protein (GAP-43) is present in plasma membranes of rat astrocytes.

“…In contrast, astrocytes appear to shut off GAP-43 expression in culture. This would explain the inability to detect GAP-43 mRNA (29) in cultured neonatal astrocytes (30) and the inability to detect GAP-43 protein in cultured embryonic astrocytes by pulsechase methods (28).…”

Opioid Signal Transduction in Intact and Fragmented SH‐SY5Y Neural Cells

“…The SH-SY5Y cells (passage 79-85) were grown for 7-20 days in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, in tissue culture flasks or 35-mm-diameter polystyrene plates as described (Fisher and Snider, 1987). In all experiments with intact cells, their viability was ascertained by measuring the cellular K+/Na+ ratio (Fischel and Medzihradsky, 1985). Under all experimental conditions a ratio of 2 3 was obtained, indicative of intact cells with an unperturbed plasma membrane (Medzihradsky and Marks, 1975).…”

Effects of extracellular sodium on μ-opioid receptors coupled to potassium channels coexpressed in Xenopus oocytes

“…To simulate recording conditions, they were incubated in batches of nine or ten oocytes in either the sodium or the choline-containing recording medium above for 2-3 h at 20-22C, washed three or four times with distilled water, carefully dried by a fine-tipped pipette, and weighed on an analytical balance. After they were homogenized at 1:100 (w/v) in 1.5 mM CsCl (the internal standard for the flame photometer), concentrated HNO 3 was added to give a final concentration of about 0.1 M, and the homogenate was incubated for 1 h at 70C [7]. The samples were centrifuged at 20,000 g for 15 min, and the supernatants were filtered (0.2 mm) and assayed for sodium and potassium in a flame photometer (Instrumentation Laboratory model 643; Lexington, Mass., USA).…”