Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Abstract

We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.5≅10 or 30 μm, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.5≤2.5 μm. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 μm) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 μm), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 μm Cch. Another muscarinic agonist, oxotremorine-M (100 μm; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (∼75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 μm) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 μm, nifedipine at 1 μm), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (∼70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 μm Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 μm) elicited a smaller change in IP₃ formation (∼25%), similar to that seen with 100 μm Oxo-M (∼20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.