Regulation of fluid-phase endocytosis in alveolar macrophages

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.

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

“…An alternate, non-concentrating transport mechanism is pinocytosis, which delivers droplets of the extracellular fluid to the endosomal apparatus in alveolar macrophages (Pataki et al, 1995). As evaluated using dextran uptake, the PMNLs are the most active cells in this respect in among several tested cell types (including the myeloid cell lines PLB 985 and U937 with K M values for quinacrine transport similar to that of lymphocytes, data not shown), with a characteristic subcellular granular distribution similar to that of quinacrine (compare Fig.…”

“…Fluid phase endocytosis (pinocytosis) was evaluated in pure population of leukocytes or myeloid cell lines by measuring the uptake of dextran (40 kDa) conjugated to rhodamine B (SigmaAldrich; final concentration in HBSS 1 mg/ml; adapted from Pataki et al, 1995). After a 30-min incubation at 37°C, the cells were centrifuged and washed prior to being photographed (green fluorescence and transmission, 1000×).…”

“…Pinocytosis (fluid phase endocytosis) is sizeable in some phagocytes and facilitates entry of extracellular fluid into the endosomal cell compartment (Pataki, 1995).…”

Alveolar Epithelial β2-Adrenergic Receptors

“…In contrast to the beneficial effects of b 2 -agonists on the alveolar epithelium, activation of b 2 AR (94) has an inhibitory effect on the function of alveolar macrophages where they diminish motility (95) via increased levels of cAMP, which has also been reported to result in diminished fluid phase endocytosis (96) and phagocytosis function (97). There is a great body of experimental data indicating that specific and nonspecific b-agonists enhance AFC in experimental models of cardiogenic and noncardiogenic pulmonary edema (1, 9, 61).…”

Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin

“…It has been demonstrated that macrophages express a small number of P2X4 at the cell surface [13,14,28] with a normalised mean current density of only 1-2 pA/pF [13]. Macrophages are known to have high rates of endocytosis [29] and rapidly turn over their repertoire of cell surface receptors. Expression of dynamin 2 in macrophages [25] may contribute to the limited surface expression of P2X4.…”