PKC and cAMP positively modulate alkaline‐induced exocytosis in the human mast cell line HMC‐1

Abstract

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used Gö6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.

Cinnamaldehyde derivatives inhibit degranulation and inflammatory mediator production in rat basophilic leukemia cells

“…Immunoglobulin E (IgE) is an activator of mast cells [5]. The aggregation of IgE receptors (FcεRI) on mast cells triggers complicated biological reactions, such as enhancement of calcium influx, rearrangement of the cytoskeleton, degranulation of prestored granules, and activation of enzymes such as protein kinase C (PKC), cyclooxygenase-2 (COX-2), 5-lipooxygenase (5-LO), and mitogen-activated protein kinases (MAPKs) [6][7][8][9]. After the activation of intracellular signaling pathways, mast cells release various inflammatory mediators, such as prestored histamine and proteases, de novo-synthesized cytokines, chemokines, and eicosanoids, such as prostaglandins and leukotrienes [10,11].…”

The effect of rottlerin in calcium regulation in HMC‐1560 cells is mediated by a PKC‐δ independent effect

“…In previous studies it was observed that inhibition of PKC-s with rottlerin (5 mM) resulted in a dramatic reduction (or even abolition) of ionomycin-induced Ca 2þ entry in HMC-1 560 cells, as Figure 1A shows [Pernas-Sueiras et al, 2006a]. In the present article it was checked if rottlerin also inhibits thapsigargininduced Ca 2þ entry.…”

“…In an earlier work [Pernas-Sueiras et al, 2006a], we reported the striking result of a total ablation of ionomycin-induced calcium increase in HMC-1 560 caused by rottlerin, but this mechanistic effect remains to be explained. In the present study, the rottlerin effects in HMC-1 560 calcium signaling, and the possible differences between ionomycin and thapsigargin-induced calcium entry through SOC channels were investigated.…”

Cross-talks between c-Kit and PKC isoforms in HMC-1560 and HMC-1560,816 cells. Different role of PKCδ in each cellular line

“…Taking into account that PKC has a central role in mast cell activation and degranulation, these results might indicate some specific role of PKCd in HMC-1 560,816 cell line activation that is not present in the other cell line HMC-1 560 [49,[53][54][55][56]. On the contrary, nuclear PKCd translocation is closely related with cell death in HMC-1 560 cells.…”