Primary cell cultures from murine kidney and heart differ in endosomal pH

Abstract

Endosomal and lysosomal pH values have been determined for many established cultured cell lines of different origins. These cell lines may be grouped into two classes based on observed differences in pH of early (recycling) endosomes. Members of the first class typically have an early endosomal pH of 6.2, whereas members of the second class typically have an early endosomal pH of 5.4. Because established cell lines may have developed artificial differences in endosomal pH due to extended culture, it remains to be determined if endosomal pH differences exist in vivo and whether they are functionally significant. To address this question, we generated adherent primary explants from mouse kidney (primarily epithelial cells) and heart (primarily fibroblasts and cardiac muscle cells). Interestingly, enhanced acidification was observed in heart cell endosomes (pH = 5.5) compared with kidney cell endosomes (pH = 6.0). These results indicate that differences in endosomal pH do not solely arise from extended cell culture and imply that such differences may be important for the proper functioning of different cell types.

Endocytic Pathway in Mouse Cardiac Cells.

“…In the first, we followed the uptake of fluorescent dextran conjugates as probe of fluidphase endocytosis at 37°C. When CM were incubated with FITC-Dx for a short time point (5 min) typically required for materials to reach the early endosomal compartments (Rybak and Murphy, 1998), we noted a punctuate fluorescence pattern: small vesicles containing FITC-dextran scattered throughout the cell's cytoplasm, mostly accumulated in the periphery of the CM presumably corresponding to early endosomes ( Fig. 2A).…”

“…A recent study showed that an increased expression of Rab1 GTPase in myocardium distorts sub-cellular localization of proteins and is sufficient to cause cardiac hypertrophy and failure (Wu et al, 2001). An enhanced acidification was observed in heart cell endosomes (pH 5.5) compared with kidney cell endosomes (pH 6.0) (Rybak and Murphy, 1998) suggesting that differences in endosomal pH may be important for the proper functioning of different cell types. Using rat cardiac myocytes it was demonstrated that the endocytic traffic is blocked in multivesicular carrier endosomes at and below 26°C, and that reduced temperature slows down transport in the recycling pathway, without a complete block (Punnonen et al, 1998).…”

“…For fluorescent approaches, the primary cultures of cardiomyocytes were allowed to take up 5 mg/ml -1 FITC-or rhodamindextran (FITC-Dx and TRITC-Dx, respectively) for 3 min at 37°C (5% CO2 incubator), extensively washed at 4°C and then further chased for 5 and 60 min at 37°C in FITC-Dx free culture medium, as previously described (Rybak and Murphy, 1998). The samples were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.2% Triton-X 100 in PBS for 2 to 3 minutes.…”

The effects of time, temperature, and pH on the stability of PDU bacterial microcompartments

“…An MCP-based delivery system can exploit the lowered pH in maturing endosomes, which reaches pH 5, to disassemble and release its cargo. [30][31][32] Coupled with encapsulated cytolysin proteins, the now-released therapeutic cargo could escape the endosome and release into the cytoplasm of target cells. It is interesting that TEM images of partially disrupted MCPs, either by high temperature or extreme pH, show that MCP bodies persist, though they are misshapen and/or smaller in size than untreated MCPs.…”

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“…Moreover, the new cell lines that support the effective replication of the human influenza viruses and have low endosomal pH are promising candidates for influenza vaccine production. Cultured mouse heart cells could be considered as an example, since they have more acidic endosomes (pH 5.5) than cultured mouse kidney cells (pH 6.0) [69].…”