Second messenger‐activated calcium influx in rat peritoneal mast cells.

Abstract

1. To study the regulation of calcium influx in non‐excitable cells, membrane currents of rat peritoneal mast cells were recorded using the whole‐cell patch‐clamp technique. At the same time, intracellular calcium concentration ([Ca2+]i) was monitored via the fluorescent calcium‐indicator dye Fura‐2, which was loaded into cells by diffusion from the patch pipette. 2. Stimulation of mast cells with secretagogues, such as compound 48/80 or substance P, caused release of Ca2+ from internal stores. In addition, external agonists also induced influx of external calcium in 26% of the cells investigated. The agonist‐stimulated Ca2+ influx was increased during membrane hyperpolarization and was associated with small whole‐cell currents. 3. Likewise, internal application of inositol 1,4,5‐trisphosphate (Ins1,4,5P3:0.5‐10 microM) elevated [Ca2+]i due both to release of Ca2+ from internal stores and to influx of external calcium. The Ins1,4,5P3‐induced influx was greater at more negative membrane potentials, suggesting that Ins1,4,5P3 opened a pathway through which calcium could enter at a rate governed by its electrochemical driving force. 4. Inositol 1,3,4,5‐tetrakisphosphate (Ins1,3,4,5P4) did not induce Ca2+ influx by itself nor did it facilitate or enhance Ins1,4,5P3‐induced Ca2+ entry. Calcium influx was also induced by inositol 2,4,5‐trisphosphate. Since this inositol phosphate is a poor substrate for Ins1,4,5P3 3‐kinase it seems unlikely that Ins1,3,4,5P4 plays a role in the regulation of the Ca2(+)‐influx pathway in mast cells. 5. The Ins1,4,5P3‐induced Ca2+ influx was associated with whole‐cell currents of 1‐2 pA or less, with no channel activity detectable in whole‐cell recordings. The small size of the whole‐cell current suggests either that the Ins1,4,5P3‐dependent influx occurs via small‐conductance channels that are highly calcium specific or that the influx is not via ion channels. 6. Agonist stimulation also activated large‐conductance (ca 50 pS) cation channels, through which divalent cations could permeate; thus, these channels represent a second pathway for Ca2+ influx. The slow speed of activation of the channels by agonists, their activation by internal guanosine 5'‐O‐(3‐thiotriphosphate) (GTP‐gamma‐S), and the inhibition of agonist activation by internal guanosine 5'‐O‐(2‐thiodiphosphate) (GDP‐beta‐S) all suggest that the 50 pS channels are regulated by a second messenger and/or a GTP‐binding protein. The activity of the 50 pS channel in mast cells is not sensitive to either Ins1,4,5P3 or Ins1,3,4,5P4. Activity of the channel was inhibited by elevated [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)