Significance of Atp-Splitting Activity of Rat Peritoneal Mast Cells in the Histamine Release Induced by Exogenous Atp

Abstract

It has previously been reported that exogenous adenosine-5'-triphosphate (ATP) evokes a marked histamine release from isolated rat mast cells with accompanying morphologi cal changes including degranulation when Cat<SUP>2+</SUP> is present in the medium (1, 2). Since it has been suggested that the energy requiring process is involved in the histamine release (3, 4), it seems to be reasonable to assume that ATP can be utilized as a source of energy supply in the process of histamine release induced by this compound, and that Ca<SUP>2+</SUP> plays a role as a cofactor of ATPase of mast cell membrane. Diamant and Kr&uuml;ger (5, 6) and Diamant (7) MATERIALS AND METHODSPreparation of cell fractions. The peritoneal fluid of Wistar rats (250-300 g, males), obtained as described before (2), was placed gently over the two layers of gum arabic solu tions of different specific gravities, 1.060 and 1.070, which were dissolved in 0.9' NaC1 in a conical centrifugation tube. Centrifugation was made at 650 for 10 minutes.Mast cells were precipitated beneath the lower layer of gum arabic. They were collected with a pipette and used as the mast cell fraction. The other cells were concentrated in the interphase between the upper and lower layers. They were mononuclear cells, lym phocytes, granulocytes and a few mast cells, of which more than 80",0 were mononuclear cells and lymphocytes.Hereafter the suspension of these mixed cells is referred to as the macrophage fraction.The mast cells and macrophages (cells of macrophage frac tion) obtained from 10-15 rats were washed 3 times with 6 nil of the incubation medium described below and resuspended in the same medium and used for each experiment. The mast cells in samples were counted in a hemocytometer after staining with toluidine blue.The protein contents of the cell fraction were determined by the method of Lowry et al. (8).

Activation and inhibition of calcium‐dependent histamine secretion by ATP ions applied to rat mast cells.

“…In the work of Dahlquist & Diamant (1974), the normal working range of ATP(total) was 20-50AM, and the concentrations of the divalent cations were reduced accordingly. We would suggest that these ATP levels may have declined during incubation, in contact with the ecto-ATPase activity which is present on the surfaces of mast cells (and on macrophages which are a common contaminant of mast cells purified by density gradient centrifugation (Sugiyama, 1971 (Chakravarty & Echetebu, 1978). Consequently the concentration of the effective free ATP may have been sufficiently labile in contact with mast cells to change during the extended times of these incubations.…”

“…Taking the purified Ca2+-pump protein (Ca2+-ATPase) of sarcoplasmic reticulum as an example, the affinity for Mg.ATP2-is 042.1061. mole-1 whereas binding of ATP4-is reported to be unmeasurable (Meissner, 1973). The activation process of mast cells is characterized by an unusually high specificity for ATP: out of several other nucleoside triphosphates which have been tested, only 2-deoxyATP had any effect on mast cell activation or inhibition (Sugiyama, 1971 a;Diamant, 1968;Cockcroft & Gomperts, 1979b). By contrast, most of the nucleoside triphosphates and even some non-nucleotides (p-nitrophenylphosphate, acetylphosphate) will support the Ca2+-ATPase of sarcoplasmic reticulum (Hasselbach & Suko, 1974).…”

“…ATP-induced anion permeability is not accompanied by loss of intracellular proteins (lactate dehydrogenase) nor does it lead to lysis, but loss of nucleotides could provide a sufficient explanation for the self inhibition by ATP of ATP-induced secretion, and of secretion induced by compound 48/80 (Diamant et al 1974) and ionophore A23187 as reported here. Also it provides an explanation for the inhibition by ATP of mast cell glycolysis (Peterson & Diamant, 1974) and for the swelling of mast cells which have been treated with ATP (Sugiyama, 1971 a).…”

“…External application of ATP to metabolically competent mast cells in the presence of Ca2+ induces exocytotic degranulation and release of histamine (Keller, 1966;Diamant & Kruger, 1967;Sugiyama, 1971 a;Dahlquist & Diamant, 1974). The original rationale for applying ATP to mast cells appears to have been generated as a consequence of some confusion arising from notions concerning the role of 0022-3751/79/6160-0066 $01.50 © 1979 The Physiological Society S. COCKCROFT AND B. D. GOMPERTS ATP in energy or phosphoryl group transfer reactions (Diamant & Kruger, 1967).…”

Release of Histamine from Rat Mast Cells by Lysosomal Cationic Proteins

“…The specific activities of these cells, measured in two experiments, were 63.6 and 63.9 nmol phosphate per 105 macrophages per 30 min. Because of this greater activity in macrophages, also noted by Sugiyama [37], only mast cell preparations of greater than 95% purity were used.…”