INTRACELLULAR CA2+ RESPONSES INDUCED BY ACETYLCHOLINE IN THE SUBMUCOSAL NASAL GLAND ACINAR-CELLS IN GUINEA-PIGS

We examined intracellular Ca2+ responses of the nasal gland acinar cells to clarify cellular responses and molecular events with regard to the regulatory mechanism of the nasal secretion. The acinar cells of the serous gland in the nasal septum of guinea pigs were obtained by meticulous and selective dissection with minimal contamination by epithelial lining cells after collagenase treatment. The dispersed acini were incubated in the oxygenated solution supplemented with fura 2 acetoxymethyl ester, and the intracellular Ca2+ concentration ([Ca2+](i)) was measured by fluorescence ratio imaging microscopy. The application of acetylcholine (ACh) to the cells induced an initially rapid increased [Ca2+](i) followed by a sustained plateau. The increase in [Ca2+](i) induced by ACh was concentration dependent, ranging between 10(-8) and 10(-4) M. The [Ca2+](i) response was completely inhibited by atropine, further indicating the involvement of muscarinic cholinergic receptors. Removal of external Ca2+ with addition of EGTA resulted in a transient increase without a sustained phase, and the transient increase was abolished by the intracellular Ca2+ antagonist 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, indicating that this increase in [Ca2+](i) was due to release from internal stores. The initial peak was not altered by changes in external pH, addition of adenosine 3',5'-cyclic monophosphate (cAMP), nor addition of phorbol 12-myristate 13-acetate (PMA) but was augmented by external K+-induced depolarization, suggesting that the transient increase was due to a change in the binding affinity to inositol 1,4,5-trisphosphate. The sustained Ca2+ entry induced by ACh was inhibited by Ni2+, but not by nifedipine. Increase in the chemical gradient for Ca2+ resulting from high Ca2+ solution in the bath enhanced the Ca2+ entry, whereas the membrane depolarization induced by external high K+ inhibited the Ca2+ influx. Decreasing extracellular pH from 7.4 to 6.4 had an inhibitory effect on the sustained [Ca2+](i) plateau. On the other hand, external alkalinization from 7.4 to 8.4 facilitated the sustained phase. Addition of cAMP or PMA did not alter the Ca2+ entry process. These results indicate that the Ca2+ entry is modulated by external pH and electrochemical gradient for Ca2+ across the membrane, but not by protein kinase A or C pathways.