ACTIVATION OF PTDINS(4,5)P-2-SENSITIVE PHOSPHOLIPASE-C IN RABBIT TRACHEAL EPITHELIAL-CELLS

A role for phospholipase C hydrolysis of phosphatidylinositol 4,5-bisp hosphate [PtdIns(4,5)P-2] as a mechanism of alpha(2)-adrenergic signal transduction in rabbit tracheal epithelial cells (tracheocytes) was i nvestigated in isolated cells grown in in vitro culture and prelabeled with myo-[H-3]inositol (3 mu Ci/ml) for 72 h. Breakdown of polyphosph oinositides was measured by using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinostol 4-phosphate [PtdIns(4)P], an d PtdIns(4,5)P-2. Inositol phosphates were separated by ion-exchange c olumn chromatography. The endogenous catecholamine l-epinephrine and a lpha(2)-adrenergic agonists clonidine and 1-(2,6-dichlorobenzylideneam ino)guanidin (guanabenz) produced a rapid transient accumulation of in ositol trisphosphate and inositol 4,5-bisphosphate and breakdown of [P tdIns(4)P] and PtdIns(4,5)P-2. The alpha(2)-adrenergic effects were no t blocked by the beta-adrenergic antagonist DL-propranolol or by the a lpha(1)-adrenergic antagonists prazosin and methylurapidil but were in hibited by pertussis toxin and blocked by yohimbine, an alpha(2)-adren ergic antagonist. The 50% effective concentration for guanabenz-stimul ated inositol trisphosphate generation was right shifted from 0.3 to 0 .9 mu M by yohimbine. The results provide the first demonstration of a lpha(2A)-adrenergic activation of pertussis toxin-sensitive PtdIns(4,5 )P-2-dependent phospholipase C in mammalian tracheocytes. The findings are consistent with previous observations on alpha(2A)-adrenergic-med iated activation of NaCl cotransport in these cells.