Related Experiment Videos
Calcium modulation of phorbol ester-induced alterations in murine macrophage morphology
Abstract:
The phorbol ester tumor promoter phorbol-12-myristate-13-acetate (PMA) induced mouse resident peritoneal macrophage spreading in an in vitro system in a time- and dose-dependent manner; this process was modified by agents which alter intracellular calcium metabolism. After a 35-min incubation with PMA, 50% of the macrophages were spread (as classified by at least a 2-fold increase in cell surface area). Also at 35 min, the median effective concentration for PMA induction of spreading was 1.6 ng/ml. The intracellular calcium antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate inhibited PMA-induced cell spreading with a one-half maximal inhibitory concentration of 8.0 microM. The calcium ionophore A23187 enhanced PMA-induced spreading by 30% at 0.1 to 10 nM. The histological dye ruthenium red, which purportedly increases intracellular calcium by displacing membrane-bound calcium stores, enhanced PMA-induced spreading up to 75% at 1.0 pM. The cationic chelator ethyleneglycolbis(beta-aminoethylether)-N,N-tetraacetic acid (1.8 and 3.6 mM) had no effect on PMA-induced spreading. Thus, PMA-induced spreading was independent of extracellular calcium but was modulated by agents altering intracellular calcium metabolism. Microfilament formation, a proposed mechanism of cell spreading, also depends on intracellular calcium availability. The microfilament inhibitor cytochalasin B inhibited PMA-induced spreading with a one-half maximal inhibitory concentration of 1 microM. Future experiments should investigate the hypothesis that calcium availability to the cytoskeletal elements regulates the morphological effects of PMA on macrophages.
Insights
Phorbol-12-myristate-13-acetate (PMA) promotes macrophage cell spreading, a process dependent on intracellular calcium. Agents altering calcium levels modulated PMA
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Phorbol ester tumor promoter, phorbol-12-myristate-13-acetate (PMA), is known to affect cellular processes.
- Macrophage cell spreading is a critical function in immune responses and tissue remodeling.
- Intracellular calcium is a key second messenger regulating various cellular activities.
Purpose of the Study:
- To investigate the role of intracellular calcium metabolism in PMA-induced macrophage cell spreading.
- To determine the relationship between PMA, calcium, and microfilament formation in macrophage morphology.
Main Methods:
- In vitro system using mouse resident peritoneal macrophages.
- Treatment with PMA at varying doses and time points.
- Assessment of cell spreading via microscopy and quantification of cell surface area.
- Modulation of intracellular calcium using antagonists, ionophores, and chelators.
- Inhibition studies using cytochalasin B, a microfilament inhibitor.
Main Results:
- PMA induced macrophage spreading in a time- and dose-dependent manner (50% spread at 35 min, EC50 = 1.6 ng/ml).
- Intracellular calcium antagonists and modulators (e.g., 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, A23187, ruthenium red) significantly altered PMA-induced spreading.
- PMA-induced spreading was independent of extracellular calcium but dependent on intracellular calcium availability.
- Cytochalasin B inhibited PMA-induced spreading (IC50 = 1 microM), suggesting microfilament involvement.
Conclusions:
- PMA-induced macrophage cell spreading is critically regulated by intracellular calcium metabolism.
- Microfilament formation, essential for cell spreading, is likely modulated by intracellular calcium availability.
- Further research should explore the direct link between calcium and cytoskeletal elements in PMA's morphological effects.