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Regulation of fluid-phase endocytosis in alveolar macrophages
1Department of Anesthesiology, University of Alabama at Birmingham 35233, USA.
Abstract:
We investigated whether fluid-phase endocytosis in rabbit alveolar macrophages (AM) was regulated by alterations in intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of freshly isolated AM were incubated with anionic dextrans (mol mass = 10 kDa), coupled to fluorescein isothiocyanate (FITC), at either 37 or 4 degrees C. There was a rapid increase in AM-associated fluorescence, quantified by laser flow-cytometry and video microscopy during the first hour of incubation at 37 degrees C, which was directly proportional to the amount of tracer present in the medium. In contrast, at 4 degrees C, AM fluorescence was similar to autofluorescence. Incubation of AM with forskolin (50 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 0.1 mM) increased their cAMP content by 67 +/- 2 and 52 +/- 5% (mean +/- SE; n = 4) and decreased FITC-dextran uptake by 29 +/- 4 and 31 +/- 4% (n = 3). On the other hand, incubation of AM with 0.5 mM IBMX inhibited FITC-dextran uptake by 62 +/- 4% (n = 3), without any further increase in cAMP. Incubation of AM with 0.4 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), a cell-permeable analogue of cAMP, decreased FITC-dextran uptake by 48 +/- 5% (n = 6). Pulse-chase experiments showed that the rate of FITC-dextran exocytosis was not affected by cAMP. We concluded that fluid-phase endocytosis in rabbit AM is regulated by cAMP and by an additional, cAMP-independent mechanism of IBMX.
Insights
Fluid-phase endocytosis in rabbit alveolar macrophages is regulated by cyclic adenosine monophosphate (cAMP). Increased cAMP levels, via forskolin or IBMX, reduce the uptake of FITC-dextran, indicating inhibitory regulation.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Fluid-phase endocytosis is a key cellular process for nutrient uptake and waste removal.
- Alveolar macrophages (AM) play a critical role in lung immunity and defense.
- Intracellular signaling pathways, such as cyclic adenosine monophosphate (cAMP), are known to modulate cellular functions.
Purpose of the Study:
- To investigate the role of intracellular cAMP in regulating fluid-phase endocytosis in rabbit alveolar macrophages.
- To determine if alterations in cAMP levels affect the rate of endocytosis in AM.
Main Methods:
- Rabbit AM were isolated and incubated with fluorescein isothiocyanate (FITC)-labeled dextran at 37°C or 4°C.
- Cellular cAMP levels were modulated using forskolin and 3-isobutyl-1-methyl xanthine (IBMX).
- FITC-dextran uptake was quantified using laser flow cytometry and video microscopy.
Main Results:
- FITC-dextran uptake was temperature-dependent, occurring rapidly at 37°C but not at 4°C.
- Forskolin and low-dose IBMX increased cAMP levels and significantly decreased FITC-dextran uptake.
- High-dose IBMX inhibited uptake independently of further cAMP increases, suggesting an additional regulatory mechanism.
Conclusions:
- Fluid-phase endocytosis in rabbit AM is regulated by intracellular cAMP levels.
- cAMP exerts an inhibitory effect on the endocytic process.
- An additional, cAMP-independent mechanism also influences FITC-dextran uptake, as suggested by the effect of high-dose IBMX.