This report describes the effect of histamine on phospholipase C (PLC) activity and calcium mobilization in cultured human ciliary muscle cells. PLC activity was assessed by measuring the production of inositol phosphates and intracellular calcium mobilization was assessed by Fura 2 ratio fluorometry. The stimulation of PLC by histamine was concentration dependent with an EC50 of 0.96 microM. The H1 antagonist chlorpheniramine blocked the response with an IC50 of 0.53 microM. Calcium fluorometry experiments indicated a mean basal calcium concentration of 36 nM with a 10(-4) M histamine induced mean peak value of 1132 nM followed by a gradually declining plateau phase. EC50 and IC50 (chlorpheniramine) values from histamine induced peak calcium concentrations agreed with the PLC results. Pretreatment of the cells with the PLC inhibitor U73122 at 10(-6) M completely blocked histamine induced calcium mobilization. Removal of extracellular calcium eliminated the plateau phase but not the initial calcium peak indicating that both intra and extracellular calcium sources are required for a normal response. The calcium ATPase inhibitor thapsigargin caused depletion of intracellular calcium stores and prevented a subsequent normal calcium mobilization response to histamine. Ryanodine, a release inhibitor of certain intracellular calcium stores, had no effect on the histamine induced response. The results of these experiments indicate that histamine, via an H1 receptor, activated the PLC second messenger pathway, and caused a multi-phasic mobilization of both intracellular and extracellular calcium. The entry of the extracellular calcium was shown to be dependent upon release of calcium from a ryanodine insensitive intracellular store.
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