PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1.
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.
Pernas-Sueiras
Octavio
O
Departamento de Farmacología, Facultad de Veterinaria, USC, 27002 Lugo, Spain.
Alfonso
Amapro
A
Vieytes
Mercedes R
MR
Botana
Luis M
LM
D000468:Alkalies / Q000494:pharmacology*; D000643:Ammonium Chloride / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects*; D006636:Histamine Release / Q000187:drug effects; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008407:Mast Cells / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*
D000468:Alkalies; D000643:Ammonium Chloride; D015759:Ionomycin; D000242:Cyclic AMP; D011493:Protein Kinase C