Benzene polyphosphates containing phosphate groups on one ring are Ins(1,4,5)P₃ 5-phosphatase inhibitors when evaluated against type-I Ins(1,4,5)P₃ 5-phosphatase. A novel biphenyl derivative, biphenyl 2,3`,4,5`,6-pentakisphosphate, with five phosphate groups on two rings was synthesized: It inhibited the activity of two inositol 5-phosphatases: type I and SHIP2 with Ins(1,3,4,5)P₄ as substrate. The inhibition was competitive with respect to the substrate. IC₅₀ value measured in rat hepatocytes, which contains the native Ins(1,4,5)P₃ 5-phosphatase, was in the micromolar range at 1.0 µM Ins(1,4,5)P₃ as substrate. Biphenyl 2,3`,4,5`,6-pentakisphosphate did not affect the activity of Ins(1,4,5)P₃ 3-kinase A in the 5-100 µM range. Surprisingly, experimental evidence supports an effect of biphenyl 2,3`,4,5`,6-pentakisphosphate at the level of the Ins(1,4,5)P₃ receptor. Finally, when injected into rat hepatocytes, the analog affected the frequency of Ca²⁺ oscillations in a positive or negative way depending on its concentration. At very high concentrations of the analog, Ca²⁺ oscillations were even suppressed. These data were interpreted as a dual effect of the biphenyl 2,3`,4,5`,6-pentakisphosphate on cytosolic [Ca²⁺] increases: an activation effect through an increase in Ins(1,4,5)P₃ level via Ins(1,4,5)P₃ 5-phosphatase inhibition and an inhibitory effect, which was exerted directly on the Ins(1,4,5)P₃ receptor. Thus, our data show for the first time that the frequency of Ca²⁺ oscillations in response to a Ca²⁺-mobilizing agonist can be controlled by inhibitors of type-I Ins(1,4,5)P₃ 5-phosphatase.-- Vandeput, F., Combettes, L., Mills, S. J., Backers, K., Wohlkönig, A., Parys, J. B., De Smedt, H., Missiaen, L., Dupont, G., Potter, B. V. L., Erneux, C. Biphenyl 2,3`,4,5`,6-pentakisphosphate, a novel inositol polyphosphate surrogate, modulates Ca²⁺ responses in rat hepatocytes.