Histamine activates phospholipase C in human airway epithelial cells via a phorbol ester-sensitive pathway M. RUGOLO, F. BARZANTI, D. C. GRUENERT, AND S. HRELIA Dipartmento di Biologia Ev.Sp. and Dipartmento di Biochimica G. Moruzzi, Universitb di Bologna, 40126 Bologna, Italy; Cardiovascular Research Institute, Gene Therapy Care Center, and Department of Laboratory Medicine, University of California, San Francisco, California 94143 Rugolo, M., F. Barzanti, D. C. Gruenert, and S. Hrelia. Histamine activates phospholipase C in human airway epithe- lial cells via a phorbol ester-sensitive pathway. Am. J. PhysioZ. 271 (Lung CeZZ. 2MoZ. Physiol. 15): L665-L671, 1996.-In human airway epithelial cell lines SHTEo- and CFNPESo-, histamine causes a transient elevation of intracellular free calcium concentration ( [Ca2 + 1;) detected by fura 2 fluores- cence, which is due to both release from intracellular stores and extracellular Ca2+ entry. The effect of histamine is abolished by the Ca 2--ATPase inhibitor thapsigargin. Hista- mine also stimulates inositol phosphate accumulation. Changes in [Ca’+]; and inositol phosphate production exhibit a similar dose-response relationship for histamine (maximal effect at 10Y4 M), with both phenomena being blocked by the H1 antagonist mepyramine and being insensitive to pertussis toxin treatment. The effects of histamine on phosphoinositide metabolism and [Ca”-]i are abolished by a short-term preincu- bation with phorbol ester, and this effect is reversed by staurosporine and calphostin C, suggesting a feedback regula- tion by protein kinase C. The results indicate that human airway epithelial cells contain H1 receptors coupled to phos- pholipase C through a pertussis toxin-insensitive G protein. H1 receptors; calcium fluxes; fura 2; inositol phosphates; cystic fibrosis IT IS WELL ESTABLISHED that several hormones, neuro- transmitters, and growth factors bind to specific mem- brane receptors, which in turn activate phospholipase C (PLC) and stimulate phosphoinositide hydrolysis, to produce diacylglycerol and inositol trisphosphate (IP& Diacylglycerol is a well-known activator of protein kinase C (PKC), whereas IP3 releases Ca2+ from intra- cellular stores (1). The increase in intracellular calcium concentration ([Ca’+]i) plays a pivotal role in many intracellular events, including activation of membrane K+ and Cl- currents. In particular, the Ca2+-regulated Cl- conductance is potentially very important in air- way epithelial cells from patients with cystic fibrosis (CF) (28), sirice the pharmacological amplification of Ca2+-mediated Cl- secretion has been suggested as a mean to bypass the adenosine 3’,5’-cyclic monophos- phate (CAMP&dependent Cl secretory pathway defect in CF (24,27,28). Clarke et al. (8) have previously reported that hista- mine induced Cl- secretion in primary cultures of human nasal epithelial cells through elevation of [Ca2+];. This [Ca2-]i elevation, in turn, causes activation of basolateral K+ conductance and apical Cl conduc- tance. Futhermore, Harris and Hanrahan (14) demon- strated that histamine stimulated a biphasic calcium response in a CF tracheal cell line, suggesting the involvement of both IP3-sensitive and IPs-insensitive intracellular Ca2+ pools. Histamine is released from mast cells and basophils, and its action is also relevant to the physiopathology of allergic responses in the airway epithelium. The broad range of biological effects of histamine is mediated by its binding to specific membrane receptors, classified into H1 (17)- and H2-type receptors (3). This classifica- tion is based on their sensitivity to different antago- nists. Another class of receptors, the H3, has recently been identified and localized exclusively in the nervous system (15). Both H1- and H2-type histamine receptors have been implicated in the airway reactivity; however, the Ca2+ response has been shown to be associated with stimulation of Hi receptors (8,14). In the present study, our attention was focused on the signaling mechanism involved in the response to hista- mine of two human airway epithelial cell lines: SHTEo-, from tracheal epithelium of a normal individual (12), and CFNPEgo-, from nasal epithelium of a CF patient (10). We therefore investigated whether the effect of histamine was due to binding to a classical HI-type receptor, associated with PLC, leading to stimulation of IP3 accumulation and Ca2’ mobilization. We also com- pared the effect of histamine in both cell lines. Further- more, it was determined whether the histamine re- sponse was modulated by PKC. MElTHODS Materials. Histamine, cimetidine, mepyramine, sulfinpyra- zone, phorbol 12-myristate 13-acetate (PMA), and pertussis toxin were from Sigma, St. Louis, MO. 4a-PMA and staurospo- rine were from BIOMOL Research Laboratories, Plymouth Meeting, PA. Thapsigargin and calphostin C were from Calbiochem, San Diego, CA. Fura 2-acetoxymethyl ester (AM) was from Molecular Probes, Eugene, OR. [2-3H]myoinositol was from Amersham International (Amersham, Bucks, UK). Dowex 1X8 (100-200 mesh formate form) was from Bio-Rad Laboratories, Richmond, CA. All other chemicals were of analytical grade. Cell cultures. SHTEo- and CFNPESo- cell lines were derived from tracheal epithelium of a normal individual and from nasal epithelium of a CF patient, respectively (10, 12). Cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 4 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% fetal calf serum. Measurements of [Ca2+]i and Mn2+ influx. Loading with 4 uM fura 2- AM was performed in trypsinized cells (4 X lo61 ml) for 30 min at 37°C with continuous stirring in DMEM growth medium (pH 7.4) supplemented with 2 mg/ml bovine serum albumin (BSA) and 0.2 mM sulfinpyrazone. Cells were washed and resuspended in the same growth medium and left at 15-17°C until use. Before each experiment, an aliquot of 3 X 10” cell s was centrifuged and resuspended in a Ca2+-free 1040-0605/96 $5.00 Copyright o 1996 the American Physiological Society L665