Journal articles

Hydrolysis-resistant analogues of GTP specifically stimulate the formation of [3H]inositol mono-, bis- and trisphosphates by saponin-permeabilized Swiss 3T3 cells prelabelled with [3H]inositol. Each inositol phosphate is formed largely by hydrolysis of its parent lipid and not by dephosphorylation of inositol 1,4,5-trisphosphate [(1,4,5)IP3]. Although hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is most sensitive to guanine nucleotides, hydrolysis of phosphatidyl-inositol (PI) and phosphatidylinositol 4-phosphate (PIP) is quantitatively more important.

We have compared the effects of pretreatment of Swiss 3T3 cell with pertussis toxin on the stimulation of DNA synthesis and phosphoinositide hydrolysis in response to a wide variety of mitogens. The toxin substantially inhibited the stimulation of DNA synthesis in response to a phorbol ester or various peptide and polypeptide growth factors irrespective of their ability to activate phosphoinositidase C.

D-Ins(1,4,5)P3 is now recognized as an intracellular messenger that mediates the actions of many cell-surface receptors on intracellular Ca2+ pools, but its complex and rapid metabolism in intact cells has confused interpretation of its possible roles in oscillatory changes in intracellular [Ca2+] and in controlling Ca2+ entry at the plasma membrane. We now report the actions and metabolic stability of a synthetic analogue of Ins(1,4,5)P3, DL-inositol 1,4,5-trisphosphorothioate [DL-Ins(1,4,5)P3[S]3].

An explanation of the complex effects of hormones on intracellular Ca2+ requires that the intracellular actions of Ins(1,4,5)P3 and the relationships between intracellular Ca2+ stores are fully understood. We have examined the kinetics of 45Ca2+ efflux from pre-loaded intracellular stores after stimulation with Ins(1,4,5)P3 or the stable phosphorothioate analogue, Ins(1,4,5)P3[S]3, by simultaneous addition of one of them with glucose/hexokinase to rapidly deplete the medium of ATP.

In permeabilized rat hepatocytes a maximal concentration (25 microM) of 2,5-di-(tert-butyl)-1,4-benzohydroquineone (tBuBHQ) mobilized 70% of sequestere Ca2+ and a half-maximal effect was produced by 1.7 microM tBuBHQ. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) stimulated release of about 40% of the intracellular Ca2+ stores. Combined applications of a range of tBuBHQ concentrations with a maximal concentration of Ins(1,4,5)P3 demonstrated that tBuBHQ has slight selectivity for the Ca2+ transport process of the Ins(1,4,5)P3-sensitive stores.

Ins(1,4,5)P3 is the intracellular messenger that in many cells mediates the effects of Ca2(+)-mobilizing receptors on intracellular Ca2+ stores. An Ins(1,4,5)P3 receptor from cerebellum has been purified and functionally reconstituted, but the relationship between this protein and the high-affinity Ins(1,4,5)P3-binding sites of peripheral tissues is unclear.

Ins(1,4,5)P3 is the intracellular messenger that mediates the effects of many cell-surface receptors on intracellular Ca2+ stores. Although radioligand-binding studies have identified high-affinity Ins(1,4,5)P3-binding sites in many tissues, these have not yet been convincingly shown to be the receptors that mediate Ca2+ mobilization, nor is it clear whether there are differences in these binding sites between tissues.

BACKGROUND AND PURPOSE: P2Y receptors evoke Ca(2+) signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved. EXPERIMENTAL APPROACH: Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca(2+)] in cultured ASMC evoked by each P2Y receptor subtype.