Intracellular calcium increases mediated by a recombinant human calcitonin receptor

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Abstract
Stable transfectants expressing a recombinant human calcitonin receptor respond to calcitonin via increased cyclic adenosine 3′,5′ monophosphate (cAMP, EC50 = 0.06 nM salmon calcitonin [sCT]) and a transient mobilization of intracellular calcium ([Ca2+]i) coincident with turnover of inositol phosphate (IP; EC50 = 6 nM sCT). Millimolar increases in extracellular calcium ([Ca2+]o, EC50 = 8 mM) cause a rapid elevation in [Ca2+]i after a calcitonin dose-dependent pretreatment of cells (pretreatment EC50 = 0.2 nM sCT). Cells exhibit persistent sensitivity to increased [Ca2+]o up to 3 h after hormone exposure and even after multiple cycles of increased [Ca2+]o followed by wash. Calcitonin pretreatment of cells also allows apparent influx of elevated extracellular strontium and manganese, but little or no effect is observed on addition of barium, cadmium, or lanthanum. Human amylin (100 nM) causes a rapid and transient increase in [Ca2+]i comparable to that of calcitonin; however, no significant response to increased [Ca2+]o is observed after amylin addition. Human calcitonin gene-related product (hCGRP) (300 nM) and forskolin do not increase [Ca2+]i or activate a sensitivity to increased [Ca2+]o. Nevertheless, human amylin and human calcitonin gene-related product (hCGRP) activate adenylate cyclase with EC50s of 0.7 nM and 8 nM, respectively. The calcium-channel drugs verapamil, BAY K 8644, diltiazem, and nifedipine have little effect on [Ca2+]i increases. The calcitonin-induced transient mobilization of calcium is inhibited by treatment of cells with cholera toxin or 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8); whereas, the response to subsequent increased [Ca2+]o is inhibited by lanthanum chloride (200 μM) and lower pH (6.0). These studies suggest that a recombinant human calcitonin receptor activates three unique signal transduction pathways in BHK cells. Subnanomolar calcitonin persistently activates adenylate cyclase and a novel pathway coupled to calcium influx while much higher calcitonin levels increase inositol phosphate turnover and generate a transient mobilization of [Ca2+]i stores.