A critical evaluation of the use of filipin‐permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ

Abstract

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.

Diminished in Situ Glucose-6-Phosphate Flux in Permeabilized Adipocytes From Streptozocin-Induced Diabetic Rats

“…4 demonstrate that the observed reduction in diabetic porous-cell G6P metabolism in the optimal range (40-70% porous) did not result from a subtle impairment in substrate ingress relative to control cells, because the differences also persisted in the highest range (70-95% porous). That optimal metabolic activity and cellular LDH retention occurred when <100% of the adipocytes were visually fluorescent is similar to the finding of McEwen et al (51) with trypan blue staining of permeabilized hepatocytes. As McEwen et al suggested, porous-cell G6P entry may be unrestricted despite insufficient dye accumulation for visual detection.…”

Unscrambling exit site patterns on the endoplasmic reticulum as a quenched demixing process

“…To further examine how ER morphology affects the ERES patterns, we sought for possibilities to convert the ER into a set of disjoint fragments. As a first means, we applied filipin to otherwise untreated cells (see Materials and methods), which led to a disintegration of the ER as previously described (38,50). As a result of the treatment, each cell displayed, on average, $700 ER fragments with a typical apparent area in the range of 0.6 mm 2 , i.e., they were on average markedly larger than ERESs in untreated cells (Fig.…”

Functional and morphological studies of protein transcytosis in continuous endothelia

“…Filipin has often been used as a permeabilizing agent (16) and has an ionophorous action on lipid bilayers (15,37). Thus in intact lung preparations, increased membrane permeability of any contaminant 125 I that is not associated with albumin could conceivably reduce or mask an inhibitory effect of filipin on 125 I-albumin uptake.…”