Interactions between inositol phosphates and cytosolic free calcium following bradykinin stimulation in cultured human skin fibroblasts

The inositol triphosphate (IP3) that results from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is generally accepted to be responsible for the mobilization of intracellular calcium. However, some studies suggest that low concentrations of agonists elevate cytosolic free calcium concentration ([Ca2+]i) without IP3 formation. Thus, in the present studies, a comparison of the temporal response of inositol phosphates (IP3, IP2 and IP) and [Ca2+]i to a wide range of bradykinin concentrations was used to examine the relation of these two signal transduction events in cultured human skin fibroblasts (GM3652). In addition, the effects of alterations in internal or external calcium on the response of these second messengers to bradykinin were determined. Bradykinin stimulated accumulation of inositol phosphates and a rise of [Ca2+]i in a time- and dose-dependent manner. Decreasing the bradykinin concentration from 1 microM to 0.1 microM increased the time until the IP3 peak, and when the bradykinin concentration was reduced to 0.01 microM IP3 was not detected. [Ca2+]i was examined under parallel conditions. As the bradykinin concentration was reduced from 1 microM to 0.01 microM, the time to reach the peak of [Ca2+]i increased progressively, but the magnitude of the peak was unaltered. These two second messengers were variably dependent on external calcium. Although the bradykinin-stimulated initial spike of [Ca2+]i did not depend on extracellular calcium, the subsequent sustained levels of [Ca2+]i were abolished in calcium free medium. The bradykinin-stimulated inositol phosphate formation was not dependent on the extracellular calcium nor on the elevation of [Ca2+]i that was produced with Br-A23187. These results demonstrate that bradykinin-induced IP3 formation can be independent of [Ca2+]i and of external calcium, whereas changes in [Ca2+]i are partially dependent on external calcium.