No 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.

No abstract

The Na⁺/Ca²⁺ exchanger has not been characterized in rat mast cells, although its exitstence has been previously suggested. In this work, we determine that this exchanger exists on rat mast cells and that it has an important regulatory role on the cellular function. We have studied uptake and release of ⁴⁵Ca in the presence of different external sodium concentrations and, under the same conditions, the simultaneous uptake of ²²Na and ⁴⁵Ca. The results show that uptake and release of ⁴⁵Ca in these cells are related to the concentration of sodium in the extracellular medium and that there is also a perfect coupling between ²²Na and ⁴⁵Ca fluxes. In these conditions, we evaluated the intracellular calcium levels in fura-2 loaded cells. When the extracellular sodium concentration was lower than 60 mM, we observed an increase in intracellular calcium, reaching its maximum when the extracellular medium has no sodium. Then we investigated the effect on histamine release. The ionophore A23187 elicits histamine release in rat mast cells, depending on the extracellular calcium concentration. This drug releases more histamine (up to twofold) with sodium concentrations <60 mM. In the presence of 2,4-dichlorobenzamil hydrochloride, a Na⁺/Ca²⁺ exchanger inhibitor, the release of histamine induced by the ionophore was lower than in controls in media with low external sodium, and, on the contrary, at extracellular sodium concentrations >60 mM, the histamine release was higher than in controls. In the same conditions, but when the Na⁺-K⁺ ATPase was inhibited by ouabain, and as a consequence more sodium was inside the cells, a high increase in histamine release induced by A23187 in a sodium-free medium was observed. Under the same conditions, a high increase in intracellular calcium takes place. The overall data are preliminary evidence suggesting the existence of a Na⁺/Ca²⁺ exchanger in rat mast cells with a threshold to get reversed at 60 mM external sodium, lower concentrations of this ion increasing internal calcium and producing higher histamine release.