Maximal concentrations of substance P and methacholine induced a rapid increase in [3H]inositol trisphosphate ([3H]IP3) formation. After about 1 min, the [3H]IP3 in the substance-P-treated cells ceased to increase further, whereas in the methacholine-treated cells [3H]IP3 continued to increase. Addition of methacholine to the substance-P-treated cells caused a rapid increase in [3H]IP3, whereas a second addition of a 10-fold excess of substance P had no effect. Pretreatment of cells with substance P, followed by removal of the substance P by washing, resulted in a decreased response to a second application of substance P. A similar protocol involving pretreatment with methacholine had no effect on subsequent responsiveness to substance P. Analysis of [3H]substance P binding to substance-P-treated cells indicated that the number of receptors for substance P was decreased, but the affinity of the receptors for substance P was unaffected. After substance P pretreatment, a prolonged incubation (2 h) restored responsiveness of the cells to substance P, measured as [3H]IP3 formation, and restored the number of binding sites to control values. These findings indicate that, in the rat parotid gland, substance P induces a homologous desensitization of its receptor, which involves a slowly reversible down-regulation or sequestration of substance-P-binding sites.

Addition of ATP (but not epinephrine, angiotensin II, vasopressin, or platelet-activating factor) to H-35 hepatoma cells whose cellular lipids have been pre-labelled with [3H]inositol, causes a rapid increase in [3H]inositol trisphosphate. In H-35 cells pre-incubated in the presence of 45CaZ+, ATP causes a similarly rapid release of 4sCaZ+. The concentration-effect relationships for inositol trisphosphate formation and Caz+ efflux are similar to those reported previously for differentiated hepatocytes. These results demonstrate that at least one of the Caz+-mobilizing receptors normally found on hepatocytes is functionally retained in the H-35 hepatoma cell line and thus could provide a useful model for the study of these receptor mechanisms in liver.(H-M cell) ATP receptor Ca2+-mobilizing receptor Purinergic receptor Inositol trisphosphate

No abstract

Summary. Agonist-stimulated hydrolysis of phosphatidylinosiiol (PI) in cell membranes has been proposed to lead to an increase of cytosol calciom concentration and activalion of the cellular response in certain smooth muscles and glands.A method is described which allows the rapid, reproducible measurement oE hydrolysis of phosphatidylinositol in mouse pancreas. The technique involves the in vivo labelling of the pancreas with iir.vo-[2-''H] inosilol. The majority of the label incorporated into phospholipids is in the form of PI. with only a small proportion of label in di-and iri-phosphoinositides. Tissue pieces of the labelled pancreala are incubaled in vitro in the presence or absence of secretagogues, and the PI in homogenates of these pieces is precipitated wilh trichloroacetic acid. No PI remains in the acid-soluhle supernatant, Tbis technique does not require the time-consuming extraction and chromalographic separation of lipids which has been necessary in other assays of PI hydrolysis. Using this method, we have confirmed lh,il PI brc;ikdt>virti i^ slimulaled by caibachol and chokcystokininoctapeptide in the presence or absence of exiiacellular Ca-+ . Agonist-Stimulated hydrolysis of PI was potentiated by extracellular Ca2 + , and was not dependent on agonist-activated Na' influx. This technique will facilitate the investigation of the importance of P! breakdown in stimulus-response coupling.

Metabolism of inositol 1,4,5-trisphosphate was investigated in permeabilized guinea-pig hepatocytes. The conversion of [3H]inositol 1,4,5-trisphosphate to a more polar 3H-labelled compound occurred rapidly and was detected as early as 5 s. This material co-eluted from h.p.l.c. with inositol 1,3,4,5 tetrakis[32P]phosphate and is presumably an inositol tetrakisphosphate. A significant increase in the 3H-labelled material co-eluting from h.p.l.c. with inositol 1,3,4-trisphosphate occurred only after a definite lag period. Incubation of permeabilized hepatocytes with inositol 1,3,4,5-tetrakis[32P]phosphate resulted in the formation of 32P-labelled material that co-eluted with inositol 1,3,4-trisphosphate; no inositol 1,4,5-tris[32P]phosphate was produced, suggesting the action of a 5-phosphomonoesterase. The half-time of hydrolysis of inositol 1,3,4,5-tetrakis[32P]phosphate of approx. 1 min was increased to 3 min by 2,3-bisphosphoglyceric acid. Similarly, the rate of production of material tentatively designed as inositol 1,3,4-tris[32P]phosphate from the tetrakisphosphate was reduced by 10 mM-2,3-bisphosphoglyceric acid. In the absence of ATP there was no conversion of [3H]inositol 1,4,5-trisphosphate to [3H]inositol tetrakisphosphate or to [3H]inositol 1,3,4-trisphosphate, which suggests that the 1,3,4 isomer does not result from isomerization of inositol 1,4,5-trisphosphate. The results of this study suggest that the origin of the 1,3,4 isomer of inositol trisphosphate in isolated hepatocytes is inositol 1,3,4,5-tetrakisphosphate and that inositol 1,4,5-trisphosphate is rapidly converted to this tetrakisphosphate. The ability of 2,3-bisphosphoglyceric acid, an inhibitor of 5-phosphomonoesterase of red blood cell membrane, to inhibit the breakdown of the tetrakisphosphate suggests that the enzyme which removes the 5-phosphate from inositol 1,4,5-trisphosphate may also act to convert the tetrakisphosphate to inositol 1,3,4-trisphosphate. It is not known if the role of inositol 1,4,5-trisphosphate kinase is to inactivate inositol 1,4,5-trisphosphate or whether the tetrakisphosphate product may have a messenger function in the cell.